Page 1

Handbook of Cell Analysis

Live Cell Imaging | Immunofluorescence | Chemotaxis Angiogenesis | Wound Healing and Migration Cell Culture Under Shear Stress | Image Analysis

2014 / 2015


Imaging Chambers

Cells & Reagents Immunofluorescence

Live Cell Imaging Chemotaxis Assays

Migration Assays Angiogenesis Assays

Flow Assays


p. 94

p. 85

REMOVABLE CHAMBERS

p. 72

micro-Insert 4 well Family

STICKY-SLIDES

p. 79

sticky-Slide 8 well

p. 89

p. 129

p. 110

sticky-Slide I Luer p. 89

sticky-Slide Chemotaxis 3D p. 101

PLATES

p. 89

sticky-Slide VI 0.4

12 well Chamber, removable

CELL MICROSCOPY

p. 69

Culture-Insert Family

µ-Plate 24 well

µ-Plate 96 well

REAGENTS

Torpedo DNA p. 48

Torpedo siRNA p. 47

ibiClone™

p. 49

ibiBoost™

Adeno Adenovirus Adeno Adenovirus Cloning Kit Transduction Enhancer

ibiPure™

Adeno Adenovirus Purification Kit

p. 42

soluble proteins

Fuse-It

MEMBRANE FUSION

dye

HT-1080 LifeAct ® Cell Line

TRANSDUCTION

p. 34

p. 51

TRANSFECTION

Life-Act® Adenoviral Vectors

ACTIN VISUALIZATION

p. 33

µ-Plate Angiogenesis 96 well

p. 50

p. 33

Life-Act ® Plasmids

µ-Plate 384 well


ibidi INSTRUMENTS page 15

ibidi Gas Incubation System

ibidi OPAL O2 Measurement System ibidi Heating System page 118

ibidi Pump System

page 142

Electric Cell Substrate Impedance Sensing (ECIS™)


µ-Slide 2 | 4 | 8 well p. 78

p. 68

µ-Slide 2 | 4 well Ph+ p. 80

p. 61

µ-Slide 2x9 well

p. 71

µ-Slide 18 well flat

p. 62

p. 85

p. 109

µ-Slide Chemotaxis 2D

WOUND HEALING

µ-Slide I Luer

µ-Slide VI 0.1 / III 0.1

p. 156

WimScratch –

Wound Healing Image Analysis

p. 152

p. 153

WimTube –

Tube Formation Image Analysis

p. 131

µ-Slide VI 0.4 / III 0.4

WimSprout –

Sprouting Image Analysis

p. 132

µ-Slide y-shaped

FLOW CHAMBERS

p. 130

WimTaxis –

Chemotaxis Image Analysis

ANGIOGENESIS CHAMBERS

p. 128

µ-Plate Angiogenesis 96 well

µ-Slide III 3 in 1

p. 154

WOUND HEALING CHAMBERS

Culture-Insert 24

µ-Dish 35mm, high ESS

p. 111

sticky-Slide Chemotaxis 3D

p. 101

µ-Slide Angiogenesis

µ-Dish Grid-50 | Grid-500

p. 110

p. 94

p. 100

p. 63

CHEMOTAXIS SLIDES

µ-Slide Chemotaxis 3D

12 well Chamber, removable

p. 64

µ-Dish glass bottom

p. 109

µ-Slide I

OPEN DISHES

µ-Dish Standard Bottom

Culture-Insert Family

ANGIOGENESIS

µ-Slide 2 | 4 | 8 well glass bottom µ-Slide 2 | 4 well Ph+ glass bottom

p. 84

µ-Slide VI flat

µ-Slide VI 0.4

p. 94

FLOW ASSAYS

p. 70

IF SLIDES

IMMUNOFLUORESCENCE AND CELL MICROSCOPY

p. 67

OPEN SLIDES

CHEMOTAXIS

p. 66


Table of Contents

Table of Contents LIVE CELL IMAGING . . . . . . . . 11 IN VIVO CONDITIONS ON THE MICROSCOPE . . . . . 12 ibidi Heating & Incubation Systems for Live Cell Imaging . . 15 Components of the ibidi Heating & Incubation Systems . . . . . . . 16 ibidi Heating System, Universal Fit . . . . . . . . . . . . . . . 18 ibidi Heating System, Multi-Well Plates . . . . . . . . . . . 20 ibidi Gas Incubation System . . 22 ibidi OPAL – Optical O2 Measurement System . . . . . . . 24 Extracellular O2 Monitoring – CPOx . . . . . . . . . . . . . . . . . . . . 25 Intracellular O2 Imaging – NanO2 | MM2 . . . . . . . . . . . . . 26 ACCESSORIES . . . . . . . . . . . . 28 µ-Dish Microscopy Rack . . . . . 28 µ-Slide Microscopy Rack . . . . . 28 µ-Slide Rack . . . . . . . . . . . . . . 29 µ-Slide Click Rack . . . . . . . . . . 29 Olaf . . . . . . . . . . . . . . . . . . . . . 29 FLUORESCENT MARKERS FOR ACTIN VISUALIZATION . 30 LifeAct ® . . . . . . . . . . . . . . . . . . 32 LifeAct ® Plasmids . . . . . . . . . . 33 LifeAct ® Adenoviral Vectors . . 33 HT-1080 LifeAct ® -TagGFP2 . . . 34 ibidi Services: Cell Lines . . . . 36 ibidi Services: Functional CellBased Assays . . . . . . . . . . . . . 38 ibidi Services: Viral Vectors . . 39

TRANSFECTION, TRANSDUCTION, AND MEMBRANE FUSION . . . . . . 40 Fuse-It Membrane Fusion . . . . 42 Fuse-It-Color . . . . . . . . . . . . . . 42 Fuse-It-P . . . . . . . . . . . . . . . . . 43 Fuse-It-Beads . . . . . . . . . . . . . 44 Fuse-It-B . . . . . . . . . . . . . . . . . 44 Fuse-It-L . . . . . . . . . . . . . . . . . 45 Fuse-It-T . . . . . . . . . . . . . . . . . 45 Fuse-It-MP . . . . . . . . . . . . . . . 46 ibiBoost™ Adeno Adenovirus Transduction Enhancer . . . . . . 47 ibiClone™ Adeno Adenovirus Cloning Kit . . . . . . . . . . . . . . . . 48 ibiPure™ Adeno Adenovirus Purification Kit . . . . . . . . . . . . . 49 Torpedo DNA Transfection Reagent . . . . . . . . . . . . . . . . 50 Torpedo siRNA Transfection Reagent . . . . . . . . . . . . . . . . . . 51 CELL PREPARATION & REAGENTS . . . . . . . . . . . . . . . 52 Collagen Type I . . . . . . . . . . . . 52 ibidi Anti-Evaporation Oil . . . . . 53 ibidi Freezing Medium Classic | Direct | HRM . . . . . . . 54 ibidi Mounting Medium . . . . . . 55 ibidi Immersion Oil . . . . . . . . . 55

6

µ-Dish Family . . . . . . . . . . . . . 60 µ-Dish 35mm, high . . . . . . . . . . . . . 61 µ-Dish 35mm, low . . . . . . . . . . . . . 61 µ-Dish 35mm, high glass bottom . . 62 DIC Lid for µ-Dishes . . . . . . . . 62 µ-Dish 50mm, low . . . . . . . . . . . . . 62 µ-Dish 35mm, high ESS . . . . . . . . . 63 µ-Dish 35mm, low Grid-500 µ-Dish 35mm, high Grid-500 . . . . . . 64 µ-Dish 35mm, high glass bottom Grid-50 | Grid-500 . . . . . . . . . . 65 µ-Slide 2 well | 4 well | 8 well . 66 µ-Slide 2 well Ph+ | 4 well Ph+ . . 67 µ-Slide 2 well | 4 well | 8 well µ-Slide 2 well Ph+ | 4 well Ph+ glass bottom . . . . . . . . . . . . . . 68 DIC Lid for µ-Slides . . . . . . . . . 68 sticky-Slide 8 well . . . . . . . . . . 69 µ-Slide 2 x 9 well . . . . . . . . . . . 70 µ-Slide I . . . . . . . . . . . . . . . . . . 71 micro-Insert 4 well Family . . . . 72 IMMUNOFLUORESCENCE (IF) . 74 µ-Slide VI 0.4 . . . . . . . . . . . . . . . 78 sticky-Slide VI 0.4 . . . . . . . . . . . 79 µ-Slide VI – flat . . . . . . . . . . . . 80 µ-Slide 18 well – flat . . . . . . . . 84 12 well Chamber, removable . 85 SCREENING . . . . . . . . . . . . . . 86 µ-Plate Family . . . . . . . . . . . . . 88 µ-Plate 24 well . . . . . . . . . . . . . 89 µ-Plate 96 well . . . . . . . . . . . . 89 µ-Plate 384 well . . . . . . . . . . . 89

www.

To find out more...

In addition to this catalog, comprehensive up-to-date information can be found on ibidi’s website under www.ibidi.com.

CELL-BASED MICROSCOPY ASSAYS . . . . 58

.com

®

Products & Applications

Workshops

• Find detailed product information and instructions

• Look for Practical Courses that are being run at ibidi in Germany

• Shop online (only available in selected countries) • Find background information and experimental details for cell-based assays

• Register for a live Webinar or listen to a previously recorded Webinar


FUNCTIONAL CELL-BASED ASSAYS . . . . . . 91

IMPEDANCE-BASED CELL ASSAYS . . . . . . . . . . . 137

WOUND HEALING AND MIGRATION . . . . . . . . . 92

Electric Cell Substrate Impedance Sensing (ECIS™) . . . . . . . . . . 142 ECIS Instrument Selection Guide . 143 ECIS Cultureware 8 well . . . . 144 ECIS Cultureware 96 well . . . 146 ECIS Transwell Measurement System . . . . . . . . . . . . . . . . . 146

Culture-Insert Family . . . . . . . . 94 Single Culture-Insert in a 35 mm µ-Dish . . . . . . . . . . . . . 95 Culture-Insert 24 . . . . . . . . . . . 95 25 Culture-Inserts for selfinsertion . . . . . . . . . . . . . . . . . . 95 ANGIOGENESIS . . . . . . . . . . 98

CUSTOM SPECIFIC SLIDES . 147

µ-Slide Angiogenesis . . . . . . 100 µ-Plate Angiogenesis 96 well 101

µ-Slide I Luer electrode . . . . . . . . 148

CHEMOTAXIS . . . . . . . . . . . 104 µ-Slide Chemotaxis Family . . 108 µ-Slide Chemotaxis 3D . . . . . . 109 µ-Slide Chemotaxis 2D . . . . . . 109 sticky-Slide Chemotaxis 3D . . . 110 µ-Slide III 3in1 . . . . . . . . . . . . . . . 111 CELL CULTURE UNDER SHEAR STRESS . . . . . . . . . . . 114 ibidi Pump System . . . . . . . . . 118 Components of the ibidi Pump System . . . . . . . . . . . . . . . . . . 119 KD Scientific Syringe Pumps 126 µ-Galaxy Incubator . . . . . . . . 127 µ-Slide I Luer Family . . . . . . . 128 sticky-Slide I Luer . . . . . . . . . 129 µ-Slide VI 0.1 | µ-Slide III 0.1 . . . 130 µ-Slide VI 0.4 | µ-Slide III 0.4 . . . 131 µ-Slide y-shaped . . . . . . . . . . 132 Flow Accessories . . . . . . . . . 133

Application

Application

Note 13

Flow experime

nt with the

IMAGE ANALYSIS . . . . . . . . 151 WimTube – Tube Formation Image Analysis . . . . . . . . . . . . . . . 152 WimSprout – Sprouting Image Analysis . . . . . . . . . . . 153 WimTaxis – Chemotaxis Image Analysis . . . . . . . . . . . 154 Chemotaxis and Migration Tool 155 WimScratch – Wound Healing Image Analysis . . . . . . . . . . . . . . 156 WimCounting – Cell Counting Image Analysis . . . . . . . . . . . . . 157 WimCAM – Chorioallantoic Membrane Assay Image Analysis . 158 WimCytotoxicity – Cytotoxicity Image Analysis . . . . . . . . . . . 159 WimComet – Comet Assay Image Analysis . . . . . . . . . . . . . 160 WimColony – Colony-Forming Image Analysis . . . . . . . . . . . . . . . 161

TECHNICAL ASPECTS OF MICROSCOPY . . . . . . . . 169 Optical Properties for High Resolution Microscopy . . . . . 170 Cell Culture Surfaces . . . . . . . 172 Cell Culture Geometry . . . . . . 176 Compatibility of Surfaces with Microscopy Techniques . . . . 178 ibidi SUPPORT . . . . . . . . . . . 181 ibidi Application Notes . . . . . 182 ibidi Movies . . . . . . . . . . . . . . 183 ibidi Practical Course: Chemotaxis Assays and Video Microscopy . . . . . . . . . 184 ibidi Practical Course: Cell Cultivation Under Perfusion and Live Cell Imaging . . . . . . 185 ibidi Webinars . . . . . . . . . . . . 186 ibidi Reference Database: . . . 187 Subject Index . . . . . . . . . . . . . 188 Catalog Number Index . . . . . 190

Note 13

6. Connecting

ibidi pump system

Custom Specific Flow Slides and Channels . . . . . . . . . . . . . 149

WimNeuron – Neurite Outgrowth Image Analysis . . 162 WimRetina – Retina Vessel Image Analysis . . . . . . . . . . . . . 163 WimTransfection – Transfection Efficiency Image Analysis . . . 164 WimLipid – Lipid Droplets Image Analysis . . . . . . . . . . . 165 WimAdipose – Adipose Tissue Image Analysis . . . . . . . . . . . . . . 166 WimTUNEL – TUNEL Assay Image Analysis . . . . . . . . . . . . . 167

the Slide with

the perfusion

and

set:

Place the fluidic µ-Slide I 0.6 Luer unit with the 1. General information the sterile work mounted perfusion bench and pinch The following set under using the plastic application note off the tubes near the valve clip (a). Put the combining the describes the surface, take µ-Slide I Luer ibidi pump system performance off the caps recommendations of a perfusion until with a µ-Slide and fill the reservoirson the working 0.6 assay there is a small for the usage Luer. Furthermore with medium are given. This Pull out the first of human umbilical I hump of liquid protocol can (see the small somemale Luer adapter vein endothelial be adapted for to make pictures). from the middle cells sure there your special are no air bubbles connector holding experimentaon the (HUVECs) For the setup l demands. slide tipping remaining inside. it upwards (b) you need the it cautiously Connect it to following material: as you see in • µ-Slide I 0.6 the female Luer the figures (c Luer, ibiTreat - f). • ibidi pump system - air pressure pump - fluidic unit - perfusion set, 15 cm, ID 1.6 mm (red marking) - hose clip • HUVECs • Endothelial Cell Growth Medium 2. Cell Culture Cultivate your cells according to your normal Endothelial Cell protocol. Growth Medium 10% fetal calf (PromoCell, Germany, For HUVECs we recommend serum (FCS). C-22010) supplement confluence when Always take care that the ed with starting a new cells are just period agglomerate experiment. reaching Cells that Repeat this very firmly and procedure with the cells homogenou you might encounterare confluent the second male a longer withover sly. Another a wipe difficulties (see figure g Depleted cells Luer adapter. important aspect by suspending - l). may not endure Then remove is the the overspill not use HUVEC shear stress and will be flushed fitness of the cells. at more than passage 4 (split from the surface. ratio 1:2)! Do 3. Preparation of the material Equilibrate all needed material overnight inside like slides, medium the incubator and tubing (perfusion bubbles emerging at 37°C and 5% CO . This sets) over time. 2 is essential to avoid air Place the perfusion set on the instructions and fluidic unit as fill in about 12 described in both reservoirs the ibidi pump ml of equilibrated should be equilibrated system medium (without get out the air slide). The level at 5 ml after bubbles from removing all of the tubes start predefined setup air from the tubes. a medium flow can be loaded To cycle. For this “Tutorial” and in the Pump purpose a choose “Load Control software. demo setups” Go to the menu Æ “Remove point Pressure air bubbles”. Shear stress 1) 50.0 mbar Flow rate Take care you None (no slide) While running Time span work as fast 23.4 ml/min the program, and careful as any disturbance gently flip on the air bubbles. possible. The infinite and will detach the tubes and cells are stressed if there is too on the adapters Check at much agitation. your cells on to remove the microscope It is crucial, that after connection all bubbles are Gas remaining to the tubing! gone before adapting the in the system slide to the fluidic can influence stop the flow. the flow rate unit! and in the ______________ ______________ worst case ______________ ______________ Application Application Note 13

© ibidi GmbH,

Version 3.0,

______________

______________

Helga Wagner,

© ibidi GmbH,

______________

March 18th,

______________

Note 13

______________

Prepared by

Version 3.0,

______________

2011

__

Page 1 of 8

______________

Prepared by

______________

Helga Wagner,

______________

March 18th,

______________

2011

__

Page 4 of 8

Support

Free Samples

Distributors

• Find experimental details for complex assays in various Application Notes

• Order a Free Sample to test ibidi’s µ-Slides, µ-Dishes, and µ-Plates with your experiments

• ibidi is represented in more than 30 countries

• Watch our Handling Movies • Search ibidi’s Reference Database for more than 3000 publications

• Find distributor contact details for your country

7


New Developments in ibidi’s µ-Slides and µ-Dishes

What’s New in 2014

What’s New in 2014

Cells and Reagents for Live Cell Imaging

µ-Slide 2 well Ph+ | 4 well Ph+

ibidi’s most popular µ-Slide for immunofluorescence and high end microscopy – now also available with 2 or 4 individual wells

Open µ-Slides with 2 or 4 wells and a special intermediate plate for excellent phase contrast over the entire well – no meniscus

Page 66

Page 67

µ-Slide 2 well | 4 well | 8 well glass bottom µ-Slide 2 well Ph+ | 4 well Ph+ glass bottom

µ-Slide III 0.4 A three-channel µ-Slide suitable for flow experiments and for immunofluorescence assays

Open µ-Slides with a glass bottom - suitable for TIRF and single molecule applications

Page 131

Page 68

DIC Lid for µ-Slides Suitable for use in DIC with all ibidi µ-Slides (except Channel and Ph+ versions) Page 68

Fuse-It Membrane fusion used as a novel and highly superior method of transiently incorporating various molecules and particles into eukaryotic cells

Torpedo siRNA dye

Page 42

≥ 90 % efficiency for the transient knockdown of mammalian gene expression – transfection efficiency optimized for use with ibidi’s µ-Slides and µ-Dishes Page 51

ibidi Services

ibiClone, ibiPure, ibiBoost

Customized generation of stable cell lines, adenoviral vectors, or lentiviral vectors, plus functional phenotypic cell-based assays

Fast and efficient adenovirus cloning, purification, and transduction Pages 47 - 49

ibidi Freezing Medium Classic | Direct | HRM Cell freezing medium not only for standard cell lines, but also for hybridoma cell lines, or ES / iPs cells with extremely high recovery rates Page 54

8

µ-Slide 2 well | 4 well | 8 well

Pages 36 - 39

Collagen Type I A high quality, collagen solution used for creating 3D collagen gels (e.g., in chemotaxis assays) Page 52


Heated Plate in Multi-Well Format for 4 µ-Slides Ideal for use in parallel, cell-based assays (e.g., chemotaxis assays) on motorized stages Page 17

ibidi Gas Incubation System A gas mixer that upgrades the ibidi Heating System to a complete stage top incubator – suitable for various experimental conditions (e.g., pH or hypoxia) Page 22

ibidi OPAL Optical O2 Measurement System Fast and non-invasive, real-time measurement of oxygen concentration directly in cell tissues or individual cells Page 24

ibidi Pump System:

Fluidic Unit Quad Combines four Fluidic Units into one system – operated by a single ibidi Pump Page 119

Cell or particle counting Page 157

WimCytotoxicity

WimTransfection

Cytotoxicity image analysis

Transfection efficiency evaluation

Page 159

Page 164

Automated Image Analysis

WimCounting

Quantitative Image Analysis of Various Cellular Assays

Advanced Solutions for Establishing Cell Environments on Microscopes

ibidi Heating System:

What’s New in 2014

What’s New in 2014

9


10


TRANSFECTION, TRANSDUCTION, AND MEMBRANE FUSION . . . . . . . . . . . . . . 40

ibidi Heating & Incubation Systems for Live Cell Imaging . . . . . . . . . . . . . . . . . . . . 15

Fuse-It Membrane Fusion . . . . . . . . . . . . . . . . 42

Components of the ibidi Heating & Incubation Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Fuse-It-P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

ibidi Temperature Controller . . . . . . . . . . . . . . 16 ibidi Gas Mixer for CO2 and O2 . . . . . . . . . . . . 16 Humidifying Column . . . . . . . . . . . . . . . . . . . . 16 Components ibidi Heating System, Universal Fit . 17 Components ibidi Heating System, Multi-Well Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 ibidi Heating System, Universal Fit . . . . . . . . . 18 ibidi Heating System, Multi-Well Plates . . . . . 20 ibidi Gas Incubation System . . . . . . . . . . . . . . 22 ibidi OPAL – Optical O2 Measurement System 24 Extracellular O2 Monitoring – CPOx . . . . . . . . 25 Intracellular O2 Imaging – NanO2 | MM2 . . . . . 26 ACCESSORIES . . . . . . . . . . . . . . . . . . . . 28 µ-Dish Microscopy Rack . . . . . . . . . . . . . . . . . 28 µ-Slide Microscopy Rack . . . . . . . . . . . . . . . . . 28 µ-Slide Rack . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 µ-Slide Click Rack . . . . . . . . . . . . . . . . . . . . . . 29

Fuse-It-Color . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Fuse-It-Beads . . . . . . . . . . . . . . . . . . . . . . . . . 44 Fuse-It-B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Fuse-It-L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Fuse-It-T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Fuse-It-MP . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 ibiBoost™ Adeno Adenovirus Transduction Enhancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 ibiClone™ Adeno Adenovirus Cloning Kit . . . . 48 ibiPure™ Adeno Adenovirus Purification Kit . . 49 Torpedo DNA Transfection Reagent Torpedo

siRNA

Live Cell Imaging

LIVE CELL IMAGING – IN VIVO CONDITIONS ON THE MICROSCOPE . . . . . . . . . . . . . . . 12

. . . . . . . . 50

Transfection Reagent . . . . . . . . . 51

CELL PREPARATION & REAGENTS . . . . . . . 52 Collagen Type I . . . . . . . . . . . . . . . . . . . . . . . . 52 ibidi Anti-Evaporation Oil . . . . . . . . . . . . . . . . . 53 ibidi Freezing Medium Classic | Direct | HRM . . . . 54 ibidi Mounting Medium . . . . . . . . . . . . . . . . . . 55 ibidi Immersion Oil . . . . . . . . . . . . . . . . . . . . . 55

Olaf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 FLUORESCENT MARKERS FOR ACTIN VISUALIZATION . . . . . . . . . . . . . . . . . . . . . . . 30 LifeAct ® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 LifeAct ® Plasmids pLifeAct-TagGFP2 | pLifeAct-TagRFP . . . . . . . 33 LifeAct ® Adenoviral Vectors rAV-LifeAct-TagGFP2 | rAV-LifeAct-TagRFP . . . 33 HT-1080 LifeAct ® -TagGFP2 . . . . . . . . . . . . . . . 34 ibidi Services: Cell Lines . . . . . . . . . . . . . . . . . 36 ibidi Services: Functional Cell-Based Assays . 38 ibidi Services: Viral Vectors . . . . . . . . . . . . . . 39

11


Live Cell Imaging – in vivo Conditions on the Microscope

Live Cell Imaging

Incubation system Gas incubation

Electric fields

Chamber geometry

Temperature

Chemical stimulation

Nutrients

Optical stimulation

pH 3D environment

CO2

Mechanical stimulation

O2

Evaporation

Volume

Surface treatment or coating

Immersion oil

Cell density

Surface

Microscopy

In vivo-like Cell Culture Conditions Cells will only behave naturally when they are cultured under the specific conditions of their biological environment. In mammals, the most prominent conditions are a temperature of 37 °C, a pH of 7.4 that is controlled by a bicarbonate buffer at a 5 % CO2 concentration, and constant concentrations of salts and nutrients. Additionally, the O2 concentration is an essential biological value. In order to achieve biologically relevant results, it is crucial to maintain these conditions on the microscope stage during live cell imaging experiments. ibidi’s product line of Heating & Incubation Systems (see page 15) allows for control of temperature, CO2 and O2 concentration, and relative humidity of live cell samples on the microscope.

Temperature Stability Temperature stability is not only important for cell culture conditions, but also very important in maintaining focus stability. The ibidi Heating Systems control temperature in an analog manner with a permanent, smooth regulation.

Temperature [°C]

37

CO2 Bicarbonate Buffer 20 0

5

10

15

20

25

30

Time [min]

H+ + HCO3-  H2CO3  H2O + CO2

The CO2 concentration in human exhaled air is 4 % to 5 %, which indicates that the CO2 dissolved in the blood is in equilibrium with air containing this CO2 concentration. Bicarbonate buffered culture media are normally designed to exhibit a pH of 7.4, at a CO2 concentration of 5 % in ambient air.

Humidity and Evaporation

12

Changing concentration

Constant concentration

The design of the µ-Slides and µ-Dishes is optimized for low evaporation. For long-term studies, evaporation can additionally be reduced through the use of silicone oil, e.g., ibidi’s Anti-Evaporation Oil (see page 53).


CO2 and O2 Control

The CO2 / O2 version of the ibidi Gas Incubation System allows you to control the oxygen in the cell culture. This function is especially beneficial, because most cells cultured in vitro live at a much lower oxygen concentration in vivo.

Inspired Air (21 kPa)

Alveolus (14 kPa)

Arterial Blood (14 kPa)

Interstitial Fluid (5 kPa) Veneous Blood (5 kPa)

Healthy Tissue (2-3 kPa)

37 °C

Measure O2

5% CO2

intra- or extracellular

90% rel. Humidity

Solid Tumor without Blood Vessel Outside (1 kPa) Inside (0 kPa)

LiveLive CellCell Imaging Imaging

When combined with the ibidi Gas Incubation System, the ibidi Heating Systems are fully functional, stage top incubators with full CO2 and humidity control.

10% O2

O2 Partial Pressure in Tissue (1 kPa = 1 % in gas mix = 7.5 mm Hg) ibidi OPAL Controller powered by Colibri

Photonics

2

A Source Light B Error

C Active Detector

Temperature Sensor

ibidi OPAL t System Optical O2 Measuremen

ibidi Heating Systems (page 15)

ibidi Gas Incubation System (page 22)

ibidi OPAL – Optical O2 Measurement System (page 24)

O2 Monitoring and Intracellular Measurement In addition to controlling the oxygen concentration in the stage top incubator, it is indispensable to know the real oxygen concentration near the cells (monitoring extracellular O2), or even inside the cells (intracellular O2). Because cells consume oxygen, the concentrations are typically much lower in cell clusters, such as tissue or spheroids. With the ibidi OPAL Optical O2 Measurement System, you can measure the real oxygen concentration directly inside the Petri dish.

The in vivo O2 concentration in most types of tissue is around 2-5 kPa, which corresponds to 2-5 % oxygen in air. Therefore, cell culture experiments at oxygen levels of 21 % are highly artificial.

NOTE The O2 concentrations are gas phase concentrations, and are specified in vol. %. NOTE: This is not an accurate unit for O2 dissolved in water and should be converted to mg/l or µmol/l, using solubility tables. However, to keep things simple, we say “the liquid contains 20% of oxygen”, which actually means “the liquid is in equilibrium with ambient air, which contains 20 vol % of oxygen”.

O2 = 10.4 %

Humidity Sensor

O2 = 8.7 %

Heated Lid Gas inlet: CO2 Humidity O2

Heated Plate

Optical O2 Measurement

The extra- or intracellular oxygen concentration is measured by beads (left) or by a cell permeable O2-sensitive fluorophore (right). See page 24 for oxygen measurement with the ibidi OPAL System.

13


Live Cell Imaging

Optimized Optical Conditions for Live Cell Imaging part from temperature stability, buffer, and humidity control, some important technical conditions A for live cell imaging also need to be considered. For optimal results in long-term assays (of 12 hours or more), condensation and focus stability are crucial parameters. The magnification of the optical system defines the resolution and the amount of cellular details that can be visualized.

Focus Stability The requirements for focus stability strongly depend on the temperature changes of the optical system and the objective’s numerical aperture (NA). The ibidi Heating & Incubation System provides crucial focus stability in live cell imaging by maintaining a continuous, smooth temperature regulation.

Condensation The independently controlled, heated glass lid of the ibidi Heating & Incubation Systems solves the problem of condensation developing in live cell imaging. By heating the lid to a temperature higher than the plate, a vertical temperature gradient is created. This gradient and an active humidity control prevent the formation of condensation on the lid of the Petri dish. The temperature at the cells’ site is maintained at a constant 37 °C. In the ibidi channel slides, condensation inside the optical pathway is intrinsically impossible. The example on the right shows this result after the sample is removed from the incubator.

ibidi solution: Heated lid

Non-heated lid 34 °C

40 °C

37 °C

37 °C

Heated plate

Heated plate

Phase contrast without condensation

Phase contrast with condensation

Condensation water 20 °C

20 °C

Light

37 °C

Open well

37 °C

Channel

Magnification and Resolution

14

Choosing the right magnification is a compromise between either having a higher resolution or more cells and statistics.

Magnification / NA

Using low-resolution microscopy with a 4x or 10x objective can be advantageous in wound healing, chemotaxis, and tube formation assays, because focussing is less delicate.

10x / 0.25

4x / 0.1

20x / 0.65 100x / 1.3

Statistics

Structural Details

Optical Stability


ibidi Heating & Incubation Systems for Live Cell Imaging

ibidi Heating System (see page 18) • Full incubator conditions on all inverse microscopes • Excellent illumination of the sample • For use with all slides, dishes, and multi-well plates

ibidi Gas Incubation System (see page 22)

Environmental Chamber

The ibidi Heating & Incubation Systems are each optimally aligned to provide perfect temperature control and the most sensitive and adjustable humidity control. The stable CO2 and O2 incubation, without evaporation, establishes physiologically relevant conditions for live cell imaging directly on the microscope.

• Upgrade to a complete stage top incubator • Stable CO2 and / or O2 incubation without evaporation

Optional: OPAL O2 Measurement System (see page 24)

Applications • Live cell imaging that requires incubator conditions on the microscope (e.g., chemotaxis, angiogenesis, cell migration, proliferation, wound healing, and cell culture under flow)

• Non-invasive, real-time measurement of oxygen concentration directly in cell tissues or individual cells

• Imaging applications with extra focus stability (e.g., TIRF, confocal microscopy)

• Fast measurement within seconds

• Anaerobic conditions or oxidative stress

Experimental Examples: Investigation of Tube Formation in Angiogenesis Research

2D and 3D Chemotaxis Experiments

Migration and Proliferation Assays 0h

0 h

12 h

2 h 6 h

HUVEC cells on Matrigel™ in a µ-Slide Angiogenesis.

Migration of a dendritic cell in a chemotactical gradient.

Closure of cell-free gap in an ibidi Culture-Insert.

15


Components of the ibidi Heating & Incubation Systems

Live Cell Imaging

ibidi Temperature Controller • Provides excellent temperature stability within the incubation chamber • Four control channels allow to connect a heated plate, a heated lid, and two additional heated components (e.g., an objective heater) Technical Features: Temperature stability

± 0.05 °C

Temperature uniformity across the insert

± 0.5 °C

Temperature range

Room temperature - 45 °C

Control channels

4

TempControl software for remote control Included and data recording External temperature sensor

Included

Adjustable alarm function

Included

Analog output for extreme noise reduction

Optional for ibidi Temperature Controller

N EW

ibidi Gas Mixer for CO2 and O2 • Precise and reliable gas incubation • Active and fast humidification without evaporation (patent pending technology) • Suitable for various experimental conditions (e.g., pH or hypoxia) • Fully compatible with the ibidi Heating Systems • Uses pressurized air to create the gas flow – no vibrations • Optional air pressure generator available (for when pressurized air is not obtainable)

Humidifying Column • Part of the ibidi Gas Incubation System • Humidifies the outgoing CO2 / O2 / air mix to minimize sample evaporation • Active humidity control inside the attached ibidi Heating System

N EW

16


Components ibidi Heating System, Universal Fit

• Can be used with any inverted microscopy system • Fits into both manual and automated stages • Can be placed into any regular multi-well frame that comes with your microscope • Adaptable to all types of microscopy chambers, such as µ-Slides and µ-Dishes plus non-ibidi formats

N EW

Heated Plate in Multi-Well Format for 4 µ-Slides

• Can be used with any inverted microscopy system • Ideal for use with motorized stages • Can be placed into any regular multi-well frame that comes with your microscope • Adaptable to all types of µ-Slides plus nonibidi formats

Heated Glass Lid, Universal Fit • For use with CO2, O2, and humidity to provide a complete environmental chamber • Independently controlled glass lid prevents the formation of condensed water on the lid of the microscopy chamber or plate • No scattering of light or shadow formation

Heated Plate with Heated Glass Bottom, Multi-Well Plates • To be used with the Nikon TI-S-E and TI-S-ER Motorized Stage which holds multi-well plates • For low magnification only (working distance > 1mm) • Suitable for DIC • For use with multi-well plates

Heated Glass Lid, Multi-Well Plates

Environmental Chamber

Heated Plate in Multi-Well Format for 1 Chamber

Components ibidi Heating System, Multi-Well Plates

• For use with CO2, O2, and humidity to provide a complete environmental chamber • Independently controlled glass lid prevents the formation of condensed water on the lid of the microscopy chamber or plate • No scattering of light or shadow formation

µ-Dish Microscopy Rack • A microscopy rack for perfect µ-Dish stabilization • Adaptable with the ibidi Heating System, MultiWell Plates • Compatible with all inverted microscopes • Lids fixed with magnetic positioning for additional stabilization

Heating Inserts • for all ibidi µ-Slides • for all ibidi µ-Dishes 35 mm • for LabTek™ / LabTek™ II chambered coverglass • To be used with the Heated Plate in Multi-Well Format for 1 chamber

Heating Insert Adapter for Perfusion Assays • To be used with the ibidi Pump System for live cell imaging under flow conditions

µ-Slide Microscopy Rack • A microscopy rack for perfect µ-Slide stabilization • Adaptable with the ibidi Heating System, MultiWell Plates • Compatible with all inverted microscopes • Lids fixed with magnetic positioning for additional stabilization

17


ibidi Heating System, Universal Fit

Live Cell Imaging

Compatible for all inverted microscopes

+ ibidi Temperature Controller

+

+

Heated Plate in multi-well format

Heated Glass Lid, Universal Fit

Heating Insert for ibidi and non-ibidi slide formats

• Excellent illumination of the sample - no condensation due to heated lid, optimal magnetic sample stabilization, and suitable for DIC

ibidi TIP Contact ibidi for a free demo of the ibidi Heating & Incubation System.

• Ideal for live cell imaging applications - full incubator conditions on the microscope • Compatible with all microscopy platforms - fits every inverted microscope that has a frame or holder for 96 well plates • Ideal for use with motorized stages when used with the Heated Plate for 4 µ-Slides

ibidi TIP The additional ibidi Gas Incubation System (see page 22) completes your heating system set-up. 0 h

12 h

24 h

Time lapse video microscopy of a rat fibroblast cell line (0 h - 60 h) Specifications:

36 h

48 h

60 h

Temperature Controller Environmental humidity

Relative humidity 0% - 99%

Environmental temperature

+5°C - +40°C (50% rel. humidity)

Dimensions (H x W x D in mm)

90 x 170 x 230

Weight

3.4 kg

Input voltage

230 V / 50 Hz 115 V / 60 Hz

Heated Plate and Lid Dimensions Plate / Lid (in mm)

127.5 x 85.5 x 8 / 135 x 85.5 x 20.5

Height of the system

26.5 mm

Weight of incubation 330 g chamber with insert

18

Gas inlet

Luer connector (M5 thread) with Luer Lock

Restrictions for objective lenses

None

Custom openings

On request

Ordering Information: Cat. No. Description Heating Systems (for use with CO2 and humidity) 10918

ibidi Heating System, Universal Fit, for 1 Chamber: ibidi Temperature Controller, Heated Plate in Multi-Well Format for 1 Chamber, 1 Heating Insert, with Heated Lid

10927

ibidi Heating System, Universal Fit, for 4 µ-Slides: ibidi Temperature Controller, Heated Plate in Multi-Well Format for 4 µ-Slides , 1 Heating Insert, with Heated Lid

10928

Heated Plate in Multi-Well Format for 4 µ-Slides

Heating Systems (Heating only) 10915

ibidi Heating System, Universal Fit, for 1 Chamber: ibidi Temperature Controller, Heated Plate in Multi-Well Format for 1 Chamber, 1 Heating Insert, without Heated Lid

Heating Inserts 10933

Heating Insert for ibidi Heating System, Universal Fit: insert for all ibidi µ-Slides

10932

Heating Insert for ibidi Heating System, Universal Fit: insert for µ-Dish 35 mm, low

10934

Heating Insert for ibidi Heating System, Universal Fit: insert for µ-Dish 35 mm, high

10937

Heating Insert for ibidi Heating System, Universal Fit: insert for LabTek™ / LabTek™ II chambered coverglass *

10936

Heating Insert Adapter for perfusion assays (to be used together with a Heating Insert for µ-Slides)

* LabTek™ is a registered trademark of Thermo Scientific.


Overview of the ibidi Heating & Incubation System, Universal Fit

ibidi Temperature Controller

Heating Inserts

Chambers

µ-Dish 35mm, high incl. glass bottom and ESS versions µ-Dish 35mm, low

Lab-TekTM / Lab-TekTM II chambered coverglass

Heated Glass Lid, Universal Fit

µ-Slide 2 well / Ph+

Environmental Chamber

ibidi Gas Mixer

µ-Slide 4 well / Ph+ µ-Slide 8 well Heated Plates in multi-well format

µ-Slide VI 0.1 / VI 0.4 µ-Slide Angiogenesis µ-Slide Chemotaxis 2D µ-Slide Chemotaxis 3D

100x

Existing stage or frame for multi-well plates on your microscope*

µ-Slide I Luer µ-Slide 18 well - flat µ-Slide 2x9 well µ-Slide y-shaped µ-Slide I

Your inverted microscope* * Not part of the ibidi Heating System. Please contact us for information on suitable microscopes.

µ-Slide III 0.1 µ-Slide III 3in1

Live Cell Imaging Under Flow Conditions The ibidi Heating & Incubation System can be adapted for live cell imaging under flow conditions. Connect the ibidi Pump System with the ibidi Heating System, Universal Fit, by using the Heating Insert Adapter for perfusion assays. This creates the perfect enivronment for long-term cell studies (several days) under flow conditions.

19


ibidi Heating System, Multi-Well Plates

Live Cell Imaging

Heating system for the Nikon TI-S-E and TI-S-ER Motorized Stage which holds multi-well plates (for low magnification only)

+ ibidi Temperature Controller

+ Heated Plate with Heated Glass Bottom

+ Heated Glass Lid, Multi-Well Plates

Multi-well Plate

• Permits the user to control the temperature of samples held in multi-well plates

ibidi TIP The additional ibidi Gas Incubation System (see page 22) completes your heating system set-up.

• Excellent illumination of the sample - no condensation due to heated top plate, and suitable for DIC • Ideal for live cell imaging applications - full incubator conditions on the microscope

Heated top plate

Heated ground plate thickness 0.7 mm

20x

Ground plate of multi-well plate

Please note: Due to the thickness of the heated ground plate, high resolution microscopy is limited.

Experimental Example: Migration and Proliferation Assay Specifications:

0h

6h

12 h

18 h

24 h

Temperature Controller Environmental humidity

Relative humidity 0% - 99%

Environmental temperature

+5°C - +40°C (50% rel. humidity)

Dimensions 90 x 170 x 230 (H x W x D in mm) Weight

3.4 kg

Input voltage

230 V / 50 Hz 115 V / 60 Hz

Heated Plate and Lid Dimensions Plate / Lid (in mm)

236 x 155 x 8 / 167 x 109 x 25

Height of the system

30 mm (22 mm from microscope stage)

Time lapse video microscopy: Closure of a cell-free gap in an ibidi Culture-Insert 24 (a 24 well plate with ready to use, individual Culture-Inserts, see page 94)

Weight of 610 g incubation chamber

20

Gas inlet

Luer connector (M5 thread) with Luer Lock

Restrictions for objective lenses

Working distance > 1 mm

Custom openings

On request

Ordering Information: Cat. No.

Description

10926

ibidi Heating System, Multi-Well Plates on a Nikon TI-S-E or TI-S-ER Motorized Stage: ibidi Temperature Controller, Heated Plate for Multi-Well Format, Heated Lid, for Nikon TI-S-E and TI-S-ER Motorized Stage


Overview of the ibidi Heating & Incubation System, Multi-Well Plates

ibidi Temperature Controller

Multi-Well Plates

µ-Plate 24 well

µ-Plate 96 well

µ-Plate Angiogenesis 96 well Heated Glass Lid, Multi-Well Plates

Any Multi-Well Plate

Adapters: Microscopy Racks

Heated Plate with Heated Glass Bottom

Environmental Chamber

ibidi Gas Mixer

µ-Dish 35mm, high µ-Dish 35mm, low µ-Slide 2 well / Ph+ µ-Slide 4 well / Ph+

Nikon TI-S-E or TI-S-ER Motorized Stage*

µ-Slide 8 well 20x

µ-Slide VI 0.1 / VI 0.4 µ-Slide Angiogenesis

Microscope*

µ-Slide Chemotaxis 2D µ-Slide Chemotaxis 3D

* Not part of this system.

µ-Slide I Luer µ-Slide 18 well - flat µ-Slide 2x9 well µ-Slide y-shaped µ-Slide I µ-Slide III 0.1 µ-Slide III 3in1

NOTE: This heating system only fits the Nikon TI-S-E and TI-S-ER Motorized Stage. For versions to fit other microscopy stages, please contact ibidi directly.

21


ibidi Gas Incubation System N

A gas mixer that upgrades the ibidi Heating System to a complete stage top incubator for all live cell imaging applications with CO2, O2, and humidity control

Live Cell Imaging

EW

+ Humidifying Column

ibidi Gas Mixer

Optional: Air Pressure Generator

• Ideal for live cell imaging applications – full incubator conditions on the microscope • Stable gas incubation without evaporation (patent pending technology) • Suitable for various experimental conditions (e.g., pH or hypoxia) • Fully compatible with the ibidi Heating Systems and stage top heaters of other suppliers

Technical Features: • Precise and reliable gas incubation for O2 and CO2 • Active and fast humidification - no evaporation • Uses pressurized air to create the gas flow – no vibrations • Optional air pressure generator available (for when pressurized air is not obtainable) Spheroid of breast cancer cells with living cells (green) and apoptotic center (red) due to oxygen depletion.

Humidity sensor

Heated lid

Location/Tissue

Gas inlet

21 kPa (=160 mm Hg)

21%

Alveolus

14 kPa (=104 mm Hg)

14%

Arterial blood

14 kPa (=104 mm Hg)

14%

Veneous blood

5 kPa (=40 mm Hg)

5%

Interstitial fluid

5 kPa (=40 mm Hg)

5%

Healthy tissue

2-3 kPa (=15- 23 mm Hg)

2-3%

1 kPa (=7.5 mm Hg) 0 kPa (=0 mm Hg)

1% 0%

2 - 11 kPa (=15 - 82 mm Hg)

2-11%

0 - 1 kPa (=0 - 7.5 mm Hg)

0-1%

20 kPa (=150 mm Hg)

20%

Normoxia conditions Hypoxia conditions

22

Corresponding O2 in Cell Culture Gas Mix (%)

Inspired air

Tumor without blood vessels Outside Center

Heated plate

Oxygen Partial Pressure p(O2)

Cell culture incubator (5% CO2) Objective lens

The ibidi Gas Incubation System provides both humid and CO2-rich air for stage-top incubators like the ibidi Heating System. The gas mixture is continuously flushed through the stage top incubator, ensuring a maximum humidity and an optimal pH for CO2-buffered liquids.

Ordering Information: Cat. No.

Description

11920

ibidi Gas Incubation System for CO2: ibidi CO2 Gas Mixer, Humidifying Column

11922

ibidi Gas Incubation System for CO2 and O2: ibidi CO2 and O2 Gas Mixer, Humidifying Column

11929

Air Pressure Generator, 1 bar, ready to use with the ibidi Gas Incubation System


Overview of the ibidi Heating & Incubation System, CO2 and O2 Control

Gas Supply (Input)

Air Pressure Generator

Air (1 bar)

CO2

N2

CO2 (1 bar)

For hypoxia only.

Lab Gas Line

Specifications:

Pressurized Air

1 bar optimum / 14.5 psi (0.8-1.2 bar / 11.6 – 17.4 psi)

CO2

1 bar optimum / 14.5 psi (0.8-1.2 bar / 11.6 – 17.4 psi)

N2

1 bar optimum / 14.5 psi (0.8-1.2 bar / 11.6 – 17.4 psi)

Tubing for all gasses (Supplied with the system)

PUR/PUN tubing 4 mm inner diameter 6 mm outer diameter 1 mm wall thickness

N2 (1 bar)

ibidi Gas Mixer

Specifications: CO2 Control CO2 Control Range 0.1% - 20% Accuracy

Environmental Chamber

Gas Supply (Input)

0.1% - 0.5% (absolute)

O2 Control O2 Control Range

0% - 21%

Accuracy

0.5 % (absolute)

Gas Flow Control Range

Gas mix, dry

Humidifying Column

Specifications: Humidity Control

 ibidi Heating System

5 - 20 l/h

Control Range

20% - 99% (Rel. Humidity)

Accuracy

1% (absolute)

Water Refill

Every 7 days

Gas mix, humidified and pre-warmed

Compatible with: • ibidi Heating System, Universal Fit (see page 18) • ibidi Heating System, Multi-Well Plates (see page 20)

23


in cooperation with

ibidi OPAL – Optical O2 Measurement System N

Versatile system for measuring the oxygen concentration in cell cultures or tissues

Live Cell Imaging

EW ibidi OPAL Controller powered by Colibri

Photonics

2

+

+

A Source Light B Error

C Active Detector

CPOx

Temperature Sensor

NanO2

ibidi OPAL ent System Optical O2 Measurem

ibidi OPAL Controller

OPAL Detector Unit, Filter Cube, and LED Light Source

Beads and nanoparticles for quantitative O2 measurement

• Exact oxygen measurement in cell cultures or tissue • Fast measurement within seconds • High spatial resolution • Non-invasive and real-time measurements • Ideal for in vitro hypoxia conditions, like 3D cultures, spheroid models, and tissue The oxygen concentration can be measured optically and directly in a 2D cell culture dish, a compact 3D cell spheroid, or even in a piece of tissue. Oxygen-sensitive beads or cell-permeable fluorophores are utilized to measure the fluorescence lifetime. Phase Contrast Image

Working Principle and Components of the ibidi OPAL System The ibidi OPAL System is easily connected to your fluorescence microscope.

Fluorescence Image

Computer

Measured Area

ibidi OPAL Controller powered by Colibri

Photonics

2

A Source Light B Error

C Active Detector

Temperature Sensor

ibidi OPAL System Optical O2 Measurement

ibidi OPAL Controller

O2 = 10.4%

O2 Concentration

24

The selected objective lens defines the measured area. An iris is used to reduce the field of measurement. The density of the CPOx beads or NanO2 labeled cells can be selected to further specify the sample area.

OPAL Detector Unit

OPAL Filter Cube

OPAL LED Light Source

Compatibility to Microscopes The ibidi OPAL System can be connected to all modern inverted microscopes from Nikon, Olympus, Leica, and Zeiss. Small adaptations need to be made, and during this process, ibidi will comprehensively support you. These adaptations include a specialized OPAL Filter Set and an LED Light Source Coupler for the microscope (included in the ibidi OPAL System). The OPAL Detector Unit fits the standard C-Mount camera port, or can also be mounted on an ocular. Note: The OPAL System measures lifetimes in the range of 1-1,000 µsec (i.e., phosphorescence). To keep things simple, ibidi uses the more common term fluorescence, instead of phosphorescence.

MM2


Extracellular O2 Monitoring

Cell Cultures

Oxygen monitoring is defined as the measurements of O2 in the direct neighborhood of cultured cells. Oxygen sensitive beads, with a diameter of 50 µm (CPOx), are used in combination with the ibidi OPAL system or a FLIM microscope to measure the oxygen concentration.

O2 = 10.4 %

Spheroids

CPOx

CPOx

• 50 µm polystyrene beads with an O2sensitive fluorophore (fluorescence lifetime) • Quantitative results with OPAL and FLIM microscopes • Biocompatible in cell culture, spheroids, or tissue • Not cell permeable • For extracellular O2 monitoring

Intracellular O2 Measurement For intracellular oxygen measurements, an oxygensensitive nanoparticle reagent is brought into the cells. The ibidi OPAL System or FLIM microscopes reveal quantitative O2 concentrations by identifying a change in the lifetime of the fluorophore NanO2.

Specifications:

CPOx orange

CPOx red

Composition

50 µm beads

50 µm beads

Ex

530 nm (510-550 nm)

540 nm (500-550 nm)

Em

600 nm (570-670 nm)

650 nm (640-670 nm)

Lifetime (37°C)

4.0 µs (21% O2) 5.3 µs (0% O2)

20 µs (21% O2) 57 µs (0% O2)

Cell Cultures

O 2 Measurement

Tissue

O2 = 8.7 %

Spheroids

Tissue

NanO2

NanO2

• For detailed information, please refer to page 27.

Ordering Information: Cat. No.

Description

74001

ibidi OPAL – Optical O2 Measurement System, Optoelectronic hardware and software for generating and processing of oxygendependent fluorescence lifetime signals: Controller, Detector Unit, LED Light Source, Adapter Set, Filter Cube, PC software

74051

Adapter Set, customized for your microscope and your requirements, for mounting OPAL to different microscopes

74101

CPOx-Beads, 50 µm, red*, fluorescence lifetime probe for extracellular O2 measurement, 3 mg (10-100 assays)

74102

CPOx-Beads, 50 µm, red*, fluorescence lifetime probe for extracellular O2 measurement, 10 mg (30-300 assays

74111

CPOx-Beads, 50 µm, orange*, fluorescence lifetime probe for extracellular O2 measurement, 3 mg (10-100 assays)

74112

CPOx-Beads, 50 µm, orange*, fluorescence lifetime probe for extracellular O2 measurement, 10 mg (30-300 assays)

74151

NanO2, fluorescence lifetime nanoparticle reagent for intracellular O2 measurement, 100 µg (10-100 assays)

* additional dyes on request

25


in cooperation with

Imaging Intracellular O2 Concentration – NanO2 | MM2 O2-Sensitive Reagents N

Nanoparticle probes for the quantitative imaging of intracellular O2 concentration (to be used with FLIM or Fluorescence Microscopes)

Live Cell Imaging

EW

+

CPOx

NanO2

FLIM

MM2 CPOx

+

NanO2

MM2

FLUORESCENCE • Monitoring in situ oxygenation of cell culture • Measuring the 3D distribution of oxygen in spheroids or tissue • Studying metabolic processes in cancer research studies • Investigating infections in cell cultures, like bacteria and fungi

Fibroblasts labeled with NanO2

Principle of FLIM – Fluorescence Lifetime Imaging Some fluorophores stay in their excited state for a relatively long time. This time period is called the fluorescence lifetime. FLIM microscopes and OPAL measure this lifetime, instead of measuring the intensity. Some long-lifetime fluorophores show a strong dependency on the surrounding oxygen level. By measuring the fluorescence lifetime, you are able to optically quantify the oxygen concentration.

23 µs 21% O2

65 µs 0% O2

In the example on the left, a 3D cell spheroid is measured optically for oxygen concentration. All cells were labeled with NanO2 and imaged with a widefield FLIM microscope. O2 concentration can be easily calculated from the fluorescence lifetime. The higher the oxygen concentration is, the shorter the fluorescence lifetime.

Overview: O2 Monitoring and O2 Imaging ibidi OPAL on Fluorescence Microscope (Fluorescence Lifetime Measurement)

CPOx Beads

NanO2

Fluorescence Microscope (Intensity-Based Imaging):

CPOx Beads

NanO2

MM2

Quantitative

Quantitative

Quantitative

Quantitative

Quantitative

Extracellular

Intracellular

Not Image-Based Not Image-Based

26

FLIM Microscope (Fluorescence Lifetime Imaging):

Extracellular

Intracellular

Intracellular

Image-Based

Image-Based

Image-Based


Intracellular O2 Imaging

Cell Cultures

For intracellular oxygen imaging, an oxygensensitive nanoparticle reagent is brought into the cells. FLIM microscopes image quantitative O2 concentrations by identifying a change in the lifetime of the fluorophore NanO2. With a standard fluorescence (or a confocal) microscope, a quantitative O2 concentration can also be determined by measuring the fluorescence intensity of the ratiometric fluorophore reagent MM2.

O2 = 8.7 %

Spheroids

NanO2

NanO2

• O2-sensitive fluorophore (fluorescence lifetime) • Quantitative results with OPAL and FLIM microscopes • Biocompatible in cell culture, spheroids, or tissue • Directly cell permeable, self-loading

O 2 Imaging

Tissue

Specifications: Composition

Nanoparticle reagent

Ex

400 nm (390-405 nm)

Em

650 nm (640-670 nm)

Lifetime (37°C)

23 µs (21% O2) 65 µs (0% O2)

• For intracellular image-based O2 measurement

MM2

MM2

• O2-sensitive fluorophore (fluorescence intensity) • Quantitative results with widefield or confocal fluorescence microscopes (independent of OPAL and FLIM systems) • Biocompatible in cell culture, spheroids, or tissue • Directly cell permeable, self-loading

Specifications: Composition

Nanoparticle reagent

Ex

400 nm (390-405 nm)

Em 1

420 nm (415-430 nm)

Em 2

650 nm (640-670 nm)

• For intracellular image-based O2 measuring 100

Intensity [a.u.]

• Ratiometric measurement based on the fluorescence intensity ratio between emission 1 (reference signal) and emission 2 (O2-sensitive signal)

80

Excitation Emission at 0% O2 Emission at 10% O2 Emission at 21% O2

0% O2 10% O2

60

21% O2

40 20

ibidi TIP The ibidi OPAL System can also be combined with NanO2, in order to measure the oxygen concentration inside living cells by analyzing the fluorescence lifetime in a non-image based manner.

0 350 400 450 500 550 600 650 700 Wavelength [nm] Emission 1 (Reference Signal)

Emission 2 (O2-sensitive Signal)

Ordering Information: Cat. No.

Description

74151

NanO2, fluorescence lifetime nanoparticle reagent for intracellular O2 measurement, 100 µg (10-100 assays)

74161

MM2, fluorescence intensity nanoparticle reagent for intracellular O2 measurement, 100 µg (10-100 assays)

27


µ-Dish Microscopy Rack

Live Cell Imaging

A microscopy rack for perfect µ-Dish stabilization

• Compatible with all inverted microscopes • Lid fixed with magnetic positioning for additional stabilization

Technical Features • Holder for up to six µ-Dishes (35 mm high or low) • Outer dimensions are the same as a multi-well plate Specifications:

Ordering Information:

Length / Width 127.5 / 85.5 mm

Cat. No.

Description

Height

19.5 mm

80035

µ-Dish Microscopy Rack with magnetic fixation, base

1

Stackable

Yes

80036

Magnetic lid for µ-Dish 35 mm, low

1

Compatible with

ibidi µ-Dishes 35 mm

80037

Magnetic lid for µ-Dish 35 mm, high

1

Material

Anodized aluminum

Sterilization

Autoclavable, alcohol

Pcs. / Box

µ-Slide Microscopy Rack A microscopy rack for perfect µ-Slide stabilization

• Compatible with all inverted microscopes • Lid fixed with magnetic positioning for additional stabilization

Technical Features • A holder for up to four µ-Slides or glass slides • Outer dimensions are the same as a multi-well plate Specifications:

28

Ordering Information:

Length / Width 127.5 / 85.5 mm

Cat. No.

Description

Height

12 mm

80030

µ-Slide Microscopy Rack with magnetic fixation, base

1

Stackable

No

80031

Magnetic lid for µ-Slides

4

Compatible with

ibidi µ-Slides Microscopy glass slides

Material

Anodized aluminum

Sterilization

Autoclavable, alcohol

Pcs. / Box


µ-Slide Rack A rack for storage and handling of µ-Slides

• No scratching of the µ-Slide bottom • Easy slide transport from incubator to work bench • Parallel handling of up to eight µ-Slides

Length / Width 215 / 90 mm

• Allows easy gas exchange through the bottom of the µ-Slides

Height

24.5 mm

Stackable

Yes

Ordering Information:

Compatible with

ibidi µ-Slides Microscopy glass slides

Material

Anodized aluminum

Sterilization

Autoclavable, alcohol

Cat. No.

Description

80003

µ-Slide Rack, for eight slides, aluminum

Pcs. / Box 1

Please note: The rack height is not for use directly with microscopes.

Accessories

Specifications:

• Ideal storage after oil immersion microscopy

µ-Slide Click Rack A rack for transport and incubation of fixed µ-Slides

• No shift, slides are fixed into position on the rack • Fits into the humidifying chamber Olaf • Parallel handling of up to four µ-Slides

Specifications:

• Outer dimensions of the rack are equal to a 96-well plate

Length / Width 127.5 / 85.5 mm

• For use mainly with µ-Slide VI and tubing Ordering Information: Pcs. / Box

Height

14.5 mm

Stackable

No

Compatible with

ibidi µ-Slides (except µ-Slide I Luer Family) Microscopy glass slides

Cat. No.

Description

80007

µ-Slide Click Rack, for four slides, plastic

1

Material

Plastic (polycarbonate)

80009

Olaf (humidifying chamber), with µ-Slide Click Rack

1

Sterilization

Alcohol

Please note: The rack height is not for use directly with microscopes and it is also not compatible with the µ-Slide I Luer Family.

Olaf A humidifying chamber for µ-Slides

• Preserves humidity, temperature, and gas concentration during the incubation of cells inside the incubator • Ideal for short-term transport of cell cultures on µ-Slides

Specifications:

• Sealed chamber for a constant atmosphere

Length / Width inside

• Inside dimensions fit multi-well format

133 / 90 mm

Length / Width outside 170 / 120 mm

• Can be used in combination with the µ-Slide Click Rack Ordering Information: Pcs. / Box

Height inside

40 mm

Height outside

53 mm

Stackable

No

Compatible with

ibidi µ-Slides Multi-well plates

Cat. No.

Description

80008

Olaf (humidifying chamber)

1

Material

Plastic

80009

Olaf (humidifying chamber), with µ-Slide Click Rack

1

Sterilization

Alcohol

29


Fluorescent Markers for Actin Visualization

Live Cell Imaging

F-Actin’s Role in Cellular Processes

Monomers Globular actin (G-actin)

The actin cytoskeleton in eukaryotes is essential to many important cellular processes, such as cell division, neuronal polarization, and cell migration. There are three components that build the cytoskeleton of eukaryotic cells: actin, microtubules, and intermediate filaments. Within the cells, actin is present in two forms. One is the monomeric, globular molecule called G-actin. G-actin has the ability to polymerize and create the second form, a double-stranded filamentous polymer called F-actin. The F-actin filaments build up different higher order structures in cells (e.g., stress fibres, lamellipodia, and filopodia). Although there are indications that G-actin plays some important roles in the cells, F-actin is primarily involved in crucial cellular processes like morphogenesis, cell division, and migration.

Polymer Filamentous actin (F-actin)

Actin Staining Techniques The successful imaging of cytoskeletal organization and dynamics can be achieved by the visualization of the actin cytoskeleton in cells or tissues using fluorescent markers.

Muscle cells

Erythrocytes Lymphocytes

Neuron

30

Epithelial cells

Before live cell imaging was available, like it is today, the only marker for F-actin was Phalloidin coupled to different fluorophores (e.g., rhodamine). Phalloidin originates from the Amanita phalloides mushroom. It is toxic because it irreversibly binds together filamentous actin, which paralyzes the cell’s cytoskeleton and leads to cell (or organism) death. Obviously, this is a disadvantage when working with living cells, and any generated data needs to be treated carefully. Phalloidin, therefore, is recommended for use only with fixed samples. Once live cell imaging became more important, and also genetically encoded fluorophores (e.g., green fluorescent protein (GFP)) became suitable for use in eukaryotic cells, researchers have been able to instantly visualize the localization and dynamics of one of these fusion proteins by coupling fluorescent proteins (FPs) to endogeneous proteins. Actin coupled with FPs, and also actin-binding proteins, have been readily used for the visualization of the cytoskeleton. However, these markers have had limitations in use such as when the FPs showed reduced functionality and altered the actin dynamics. This has also been true with other markers.


Advantages of Using LifeAct® for Actin Visualization

Live Cell Imaging Influence on Actin Dynamics Signal-to-Noise Ratio

FP-Actin

LifeAct

++

n.a.

++ (Peptide)

(Toxin) -(Toxin)

++

++

(High)

++ (None)

++

+

++

LifeAct specifically stains F-actin and perfectly co-localizes with existing markers.

In contrast to the current approaches, LifeAct visualizes F-actin within fixed and living cells, without compromising cellular processes, such as the locomotion of dendritic cells or neuronal polarization. LifeAct, a 17-amino acid peptide, is derived from a protein found in Saccharomyces cerevisiae. As a novel actin marker, LifeAct combines crucial characteristics that make it the best marker available today. To date, it is the shortest actin marker for living cells and has a low binding affinity to F-actin. Although it is high enough to label F-actin very specifically, LifeAct does not disturb actin kinetics. Because of this, actin’s functionality remains unrestricted. So, by using LifeAct for visualization of the actin cytoskeleton, researchers achieve the most authentic results. LifeAct plasmids (see page 33) allow for transient or stable expression of LifeAct for different applications. Difficult-to-transfect cells, such as primary cells, are easily transduced with the LifeAct adenoviral vectors (see page 33). A stable LifeAct-TagGFP2 expressing HT-1080 cell line (see page 34) allows long-term actin visualization without interference with cytoskeletal dynamics. Transfection or transduction of LifeAct can easily be done in ibidi’s µ-Slides and µ-Dishes. With the Torpedo Transfection Reagents, ibidi provides the ideal reagents – cationic lipids – and protocols for cell transfection. Torpedo DNA (see page 50) was optimized specifically for the transfection of mammalian cells with plasmid DNA in ibidi’s µ-Slides. Torpedo siRNA (see page 51) enables successful gene silencing experiments when using ibidi’s µ-Slides in live cell imaging.

LifeAct-GFP

Actin Visualization

Fixed Samples

Phalloidin

Co-Localization Assay

Phalloidin

Merge

ibidi MOVIE For practical details please watch our movies on www.ibidi.com: LifeAct: Actin Marker for Live Cell Imaging (MV 19) Immunofluorescence Using the µ-Slide VI (MV 18) Cell Transfection Using Torpedo DNA (MV 24)

31


LifeAct®

Live Cell Imaging

An actin marker for the visualization of F-actin in living cells

• Brilliant visualization of F-actin in live cell imaging - perfect imaging of cytoskeletal organization and cellular dynamics • No interference with cytoskeletal dynamics - unrestricted actin functionality • Excellent signal-to-noise ratio

Applications • Cytoskeleton organization:

- Non-toxic actin visualization - Fluorescence staining

• Cytoskeletal dynamics: ibidi APPLICATION NOTE ibidi’s application note Adenoviral Transduction of Human Cells (AN 28) explains how to design an approach for transducing human cells.

ibidi MOVIE Watch our movie LifeAct: Actin Marker for Live Cell Imaging (MV 19) on www.ibidi.com.

- Actin dynamics in cellular processes - Live cell imaging and microscopy

LifeAct, a 17-amino acid peptide, is derived from a protein found in Saccharomyces cerevisiae. LifeAct stains filamentous actin (F-actin) structures in living or fixed eukaryotic cells and tissues. In contrast to GFP-actin and its alternatives such as actin-binding proteins, LifeAct does not interfere with actin dynamics in vitro and in vivo, as shown in several cell types and model organisms.* Versatile products are available for the use with LifeAct for your specific experimental needs (see page 33). Plasmids may be used in many cell types when a transient or stable expression of LifeAct is needed. In this case, researchers can choose between two different promoters, CMV (cytomegalovirus) or CAG (modified chicken β-actin). For many standard cell lines, the CMV promoter is the best choice as it leads to strong expression of LifeAct. However, when working with sensitive cells such as neuronal or embryonic stem cells, it is most often recommended to use the CAG promoter, because it does not have a viral origin and an interference with cellular defense mechanisms (“silencing mechanisms”) is less likely. Adenoviral vectors are available for difficult-to-transfect cells or when up to 100 % transgene expressing cells are needed. In addition, different stable cell lines (see page 34) are available, which help facilitate daily lab life. You can immediately start performing experiments without the time-consuming and laborious generation of stable cell lines, while still assuring high-quality standards.

LifeAct is a registered trademark of ibidi GmbH. For license and product use information please refer to our website: www.ibidi.com * Original LifeAct publication in Nature Methods: Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, Bradke F, Jenne DE, Holak TA, Werb Z, Sixt M & Wedlich-Söldner R. Lifeact – a versatile marker for the visualization of F-actin. Nature Methods 5, 605-607 (2008)

32

** Information about TagGFP2 and TagRFP at www.evrogen.com


LifeAct® Plasmids pLifeAct-TagGFP2 | pLifeAct-TagRFP An actin marker for the visualization of F-actin in living cells after plasmid transfection

• For transient and stable transfections • Superb biocompatibility - non-toxic staining of living samples After transfection of cells with pLifeAct, F-actin is visualized using the fluorescence markers TagGFP2 or TagRFP**. Stable transfection of cell lines allows long-term actin staining for various applications. Ordering Information:

Specifications / Content:

Cat. No.

Description

Pcs. / Box

60101

Plasmid p  LifeAct-TagGFP2, ready to use, 20 µg, concentration 500 ng / µl (CMV Promoter; green fluorescence)

1

60102

Plasmid pCMV LifeAct-TagRFP, ready to use, 20 µg, concentration 500 ng / µl (CMV Promoter; red fluorescence)

1

60106

Plasmid pCAG LifeAct-TagGFP2, ready to use, 20 µg, concentration 500 ng / µl (CAG Promoter; green fluorescence)

1

60107

Plasmid pCAG LifeAct-TagRFP, ready to use, 20 µg, concentration 500 ng / µl (CAG Promoter; red fluorescence)

1

CMV

Concentration

500 ng / µl

Storage

-20 °C

TagGFP2 (Exmax / Emmax)

483 / 506 nm

TagRFP (Exmax / Emmax)

555 / 584 nm

Actin Visualization

Technical Features

LifeAct® Adenoviral Vectors rAV-LifeAct-TagGFP2 | rAV-LifeAct-TagRFP An actin marker for the visualization of F-actin in living cells after adenoviral transduction

Technical Features • Access to difficult-to-transfect cells, such as primary cells • Superb biocompatibility - non-toxic staining of living samples After the transduction of cells with rAV-LifeAct, F-actin is visualized using the fluorescence markers TagGFP2 or TagRFP**. With this method, transduction efficiencies of up to 100 % can be attained, even in difficult-to-transfect cell types like primary cells (e.g., neuronal cells). Ordering Information:

Specifications / Content:

Cat. No.

Description

60121

Adenovirus rAV CMV-LifeAct-TagGFP2, ready to use, 1 x 10 9 IU, concentration 1 x 1010 IU / ml (CMV Promoter; green fluorescence)

1

Adenovirus rAV CMV-LifeAct-TagRFP, ready to use, 1 x 10 9 IU, concentration 1 x 1010 IU / ml (CMV Promoter; red fluorescence)

1

60122

Pcs. / Box

ibidi TIP Check out ibidi’s Adenovirus Transduction productline (page 47 ff.) for fast and efficient adenovirus cloning, purification, and transduction.

Concentration

1 x 1010 IU / ml

Storage

-80 °C

TagGFP2 (Exmax / Emmax) 483 / 506 nm TagRFP (Exmax / Emmax)

555 / 584 nm

NOTE: Please note that you will be working with a product containing infectious virus. Follow the recommended NIH guidelines for all materials containing Biosafety Level 2 organisms.

33


HT-1080 LifeAct®-TagGFP2

Live Cell Imaging

A stable LifeAct-TagGFP2 expressing human fibrosarcoma cell line

• Long-term actin visualization – 25 passages guaranteed • Characteristics proven to be identical to the parental HT-1080 cell line • LifeAct technology guarantees no interference with cytoskeletal dynamics

Applications • Live cell imaging of cytoskeletal organization and dynamics • Functional cell-based assays, such as chemotaxis and migration • Usable as a characterized control in your experiments

Specifications: Cells / vial

5 x 10 5

Medium

ibidi Freezing Medium

Storage

Liquid nitrogen

Biosafety Level 1

The HT-1080 LifeAct-TagGFP2 cells present highly dynamic filamentous actin. As a result of stable LifeAct expression, perfect visualization of filamentous actin with very low background fluorescence is achieved without any interference to cellular dynamics. Moreover, full characterization of the cells revealed that they displayed identical behavior to wild type (wt) cells. Due to the cell line’s fast growth and the absence of contact inhibition, it constitutes an ideal tumor model. These characteristics combined with bright and specific F-actin staining lead to a powerful tool with versatile applications in live cell imaging and high-content approaches.

Sterility

Tested for yeast and bacteria including mycoplasma

Promoter

CAG (modified chicken β-actin)

Technical Features:

Transgene

LifeAct-TagGFP2*

• • • •

* For further information on TagGFP2, please go to www.evrogen.com.

Homogeneous cell pool Assured cell line identity Purity tested Optimal transgene expression rates for microscopy assays

Organism

Homo sapiens (human)

More stably LifeAct transfected cell lines will be available in the future. Please contact ibidi for detailed information.

Source

Connective tissue disease: fibrosarcoma

Other available services include:

Origin information:

Age / Gender 35 years / Male Ethnicity

Caucasian

Growth properties

Adherent

Morphology

Epithelial

in cooperation with

• Custom stable cell pools • Clonal cell line service • Functional cell characterization Please refer to page 36 for detailed information.

Ordering Information: Cat. No.

Description

40101

HT-1080 LifeAct-TagGFP2, HT-1080 cells expressing LifeAct-TagGFP2, 5 x 10 5 cells / vial

Pcs. / Box 1

Coming soon:

www.sirion-biotech.com

34

40102

HT-1080 LifeAct-TagRFP, HT-1080 cells expressing LifeAct-TagRFP, 5 x 10 5 cells / vial

1


Basic Characterization of HT-1080 LifeAct-TagGFP2

Genomic authentication of human cell lines by means of short tandem repeat (STR) analysis is widely used to assure cell line identity and to investigate cell line purity. Short tandem repeat polymorphisms in the human genome are used to differentiate between individuals. By identifying the short repeats of a specific sequence at typical locations in the genome, it is possible to

HT-1080 wt HT-1080 LifeAct

20

98 % positive cells

Cell count (x 10 2)

Cell Number [x 105 cells]

25

create a genetic profile of an individual human being (as used in forensics), or of a cell line. ibidi’s HT-1080 LifeAct-TagGFP2 cells were analyzed using the above mentioned method. The analysis was performed by DSMZ, an ISO-certified Leibniz institute. The analysis revealed that HT-1080 LifeAct-TagGFP2 cells are identical to HT-1080 wt cells.

Actin Visualization

Genomic Characterization:

15 10 5

wt control

0 1

2

3

4

5

6

7

Time [d]

Fluorescence signal 488 nm

Cell Proliferation Analysis:

Fluorescence Intensity in Cell Population:

HT-1080 LifeAct-TagGFP2 and wt cells were cultured for 8 days using the same defined conditions as the cultures’ start points, and then analyzed for cell number each day. Results from this experiment showed that both cell lines grew at equal rates over the predefined period of time. This result was confirmed using statistical tests.

HT-1080 LifeAct-TagGFP2 and wt cells were analyzed for fluorescence intensity and number of positive cells using flow cytometry. At least 95 % of the cells were fluorescent with on average a two log shift in fluorescence intensity compared to wt cells.

Functional Characterization of HT-1080 LifeAct-TagGFP2 0.3

Cell front velocity [µm / h] 40

LifeAct

0.1

wt

FMI

0.2

30 20

0.0

10

wt

LifeAct

Chemotaxis 2D and 3D

Wound Healing:

Chemotaxis assays of HT-1080 LifeAct-TagGFP2 and wt cells were analyzed in µ-Slides Chemotaxis 2D and Chemotaxis 3D, using fetal calf serum (FCS) as a chemoattractant (see Application Notes 14 and 17 for further details). Results from these assays showed that HT-1080 LifeAct-TagGFP2 cells migrate with the same speed and forward migration index (FMI) as wt cells. These results were confirmed using the Student’s t-test. p < 0.05 was considered significant.

HT-1080 LifeAct-TagGFP2 and wt cells were seeded in a µ-Slide 8 well combined with a Culture-Insert. After 24 hours of video microscopy, wound closure was analyzed using the WimScratch automated software solution. HT-1080 LifeAct-TagGFP2 and wt cells showed comparable cell front velocities. These results were confirmed using the Student’s t-test. p < 0.05 was considered significant.

35


ibidi Services: Cell Lines

Live Cell Imaging

Customized generation of stable cell lines that are ready to use in your experiments

• Maximum flexibility in experimental design • Strong gene expression/knockdown • Delivery of validated stable cell pools in less than 7 weeks

Stable cell lines simplify a researcher’s life in many ways and are also essential tools for performing cell-based assays in a more comfortable and reliable manner. Therefore, ibidi offers the custom generation and production of recombinant stable cell lines that provide the highest flexibility for your research needs. Our service includes the cloning of an expression cassette into the according vector, the transfection/transduction of the cell line, and the selection of the stable cell pool using antibiotics. In addition, the transgene expression is verified using qRT-PCR and the cell pool is validated using standard quality control tests.

Custom Stable Cell Pools In cooperation with SIRION BIOTECH (www.sirion-biotech.com), ibidi offers the following custom stable cell pools that can be used in various applications: Bright field and fluorescence images of stably transfected Normal Human Dermal Fibroblasts (NHDF) expressing LifeAct-RFP.

LifeAct-GFP / RFP Expressing Cell Pools: These cell pools allow in vivo imaging of the actin cytoskeleton and permit the study of actin dynamics by using the advantageous LifeAct actin marker. Cell Pools for Protein Overexpression / Knockdown: These cell pools allow the stable expression of your gene of interest in your mammalian cell line of interest for use in: • Purification of proteins • Functional gene analysis • Target identification and validation

Western Blot showing the expression of two splice variants of a glycolytic enzyme in stable THP-1 cell pools (Lanes 4 and 5), both of which are not expressed in the parental cell line (lanes 3). Lanes 1 and 2 show positive controls.

36

• Generation of reporter cell lines, e.g., for screening


Cell Pools for Simultaneous Expression of Two or More Proteins: These cell pools allow stable expression of your genes of interest in your cell line of interest for use in: Analysis of protein / protein interaction Expression of heteromeric proteins Generation of reporter cell lines, e.g., for screening Coexpression of fluorescent or luminescent proteins for FACS analysis or in vivo imaging (xenotransplant)

Stable murine 4T1 cell pool simultaneously expressing RFP and GFP from two different vectors.

Services available with custom stable cell pools include: • Cloning of the cDNA from your gene(s) of interest into an expression vector • Verification of cloning success by DNA-sequencing • Transfection / Transduction of your cell line • Generation of stable cell pools by antibiotic selection • Expression quantification of your gene of interest using qRT-PCR and / or Western Blot, or fluorescence • Delivery of validated frozen cells

ibidi Services

• • • •

FACS of Stable Cell Pools If you want to improve the homogeneity of your cell pool using fluorescent or luminescent proteins, ibidi provides a cell sorting service based on flow cytometry. This additional service comprises: • FACS of a stable cell pool to isolate specific subpopulations • Customized expansion and cell banking of sorted cell pools

Histograms showing a stable HT-1080 cell line, generated using LifeAct-TagGFP2, before (middle) and after (top) FACS in comparison to HT-1080 wt cells (bottom). Through the use of FACS, a homogeneous cell population with more than 95 % of fluorescent cells was obtained.

Clonal Cell Line Service For customers who need optimal transgene expression and guaranteed stability of cell clones, ibidi provides a Clonal Cell Line Service. This additional service includes: • Establishment of single cell clones using the Genetix Clone select Imager • Small scale expansion of individual clones • Identification of „High Performers“ via expression quantification of a target gene, or gene of interest, using qRT-PCR and / or Western Blot, relative to control cells • Large scale expansion and cell banking of selected clones • Final Quality Control of transgene expression, growth characteristics, and long-term cultivation (20 passages)

Please send a Service Request to info@ibidi.com and receive detailed information and a quote.

Caco-2 cells are used as an in vitro model of the human small intestinal mucosa to predict the absorption of orally administered drugs. In order to understand mechanisms of drug efflux better, SIRION BIOTECH generated a Caco-2 cell line with stable knockdown of P-Glycoprotein 1 (pgp1) of more than 80 % over 20 passages.

in cooperation with

www.sirion-biotech.com

37


ibidi Services: Functional Cell-Based Assays N

Functional phenotypic cell-based assays that are fitted to your needs and performed by ibidi’s experts

Live Cell Imaging

EW

• Fast and reliable, high-content results

Please send a Service Request to info@ibidi.com and receive detailed information and a quote.

• High throughput performance by specialists • High-quality support for assay establishment at your site

Comprehensive Phenotyping by Functional Cell Characterization ibidi is a notable expert in the field of functional cell-based assays, with long-term hands-on knowledge as well as solution-finding experience for you, our customers. Benefit from over 10 years of ibidi’s expertise in developing and performing cell-based assays at our facility, as well as on-site technical training for establishing the assays at your own lab.

Chemotaxis in 2D and 3D

0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1

P1 0 P1 5 P2 1 P3 2 P3 5 P4 1

t

P5

w

P2

-0.2

Chemotaxis assays of a wt and stable HT-1080 cell line, over 40 passages, performed in a μ-Slide Chemotaxis 3D (see page 109). A trajectory plot and the parallel FMI values of the obtained, high-content data are shown.

Wound Healing

60

40

20

C 7

C 6

C 8

C 5

C 4

C 3

C 2

t w

C 1

0

Total tube length [µm/mm²]

Cell migration assay of epithelial cells in a µ-Plate 24 well with Culture-Inserts (see page 95). Various compounds (C1-C8) show different effects on cell front velocity.

38

Chemotaxis is a crucial mechanism in various cellular processes, such as organism development or cancer cell migration. Using our technology, we are able to provide stable conditions over long time periods, while the cells are examined by video-microscopy.

12000 10000 8000 6000 4000 2000 0

Tube formation assay of endothelial cells performed in a µ-Plate Angiogenesis 96 well (see page 101). The graph shows reproducible total tube length results in each well of the plate.

Using our technology for analyzing cells during the wound healing process is the best way to gain reproducible, highcontent data. When investigating the potency of drugs, our specialized expertise enables high-throughput experimentation with reliable results.

Angiogenesis Angiogenesis is an important process during organism development or cancer growth. Using our technology, we are able to provide high-throughput performance with a standardized and reliable approach.


ibidi Services: Viral Vectors

N

EW

Customized generation of adenoviral and lentiviral vectors that are ready to use in your experiments

• High quality and fast processing times • Expert support

Please send a Service Request to info@ibidi.com and receive detailed information and a quote.

ibidi offers the custom generation and production of recombinant adenoviral and lentiviral vectors that provide the highest flexibility for your research needs. Our service includes the cloning of an expression cassette into the viral vector, transfection of the production cell line, and amplification of virus particles. In addition, the viral particles are purified and the infectious titer is determined.

ibidi Services

• High flexibility for your experiments

Applications: • Fluorescent labeling or silencing of your protein of interest

rAV-LifeAct virus particle

• Constitutive or inducible expression of transgene • Generation of stable cell lines for long-term expression (only lentiviral vectors)

CAR

• High efficiency, also in difficult-to-transfect cells (e.g., neurons) or terminally differentiated cells (e.g., neutrophiles)

Cell membrane

ENDOCYTOSIS

LifeAct protein TRANSLATION

Endosome

Adenovirus Generation Recombinant adenoviruses are tools that are widely used in research and also in therapeutic applications. Due to their broad host tropism, they are ideal vectors for transferring genes into the majority of mammalian cells. Adenoviral vectors are the perfect tools for the high-level expression of recombinant proteins (e.g., rAVLifeAct Adenoviral Vectors, see page 33), and they also mediate very efficient gene knockdowns. Adenoviral vectors are episomal vectors that do not integrate into the host genome.

mRNA RELEASE OF DNA

TRANSCRIPTION

Transduction of mammalian cells using an adenoviral vector, here rAV-LifeAct (see page 33)

lentiviral particle

Lentivirus Generation Recombinant lentiviral vectors have been shown to be powerful tools for stable gene transfer to both dividing and non-dividing cells in vitro and in vivo as they integrate into the host genome. They have a broad host cell range that also includes cell types such as neurons, lymphocytes, and macrophages. Moreover, lentiviral vectors have also proven to be effective in transducing brain, liver, muscle, and retina in vivo without toxicity or immune responses.

FUSION

Cell membrane

RELEASE OF RNA

ssRNA

LifeAct protein TRANSLATION

REVERSE TRASCRIPTION

Nucleus

mRNA

dsRNA

TRANSCRIPTION

dsDNA REVERSE TRASCRIPTION

STABLE INTEGRATION

Fusion of a lentiviral vector with the cell membrane of mammalian cells

39


Transfection, Transduction, and Membrane Fusion

Live Cell Imaging

When analyzing biological coherences, such as protein interactions or signal transduction in eukaryotic cells, it is often necessary to transfer genetic material or other compounds, e.g., proteins, into cells.

Transfection pLifeAct plasmid

Transfection reagent

DNA lipid complex Cell membrane

ENDOCYTOSIS

LifeAct protein TRANSLATION

mRNA

Endosome

RELEASE OF DNA

TRANSCRIPTION

Transfection of eukaryotic cells with plasmid DNA, here pLifeAct (see page 33)

rAV-LifeAct virus particle

CAR

With the Torpedo Transfection Reagents, ibidi provides the ideal reagents – cationic lipids – and protocols to successfully perform transfection experiments directly in the ibidi µ-Slides. Torpedo DNA (see page 50) was optimized specifically for the transfection of mammalian cells with plasmid DNA in ibidi’s µ-Slides. Torpedo siRNA (see page 51) enables successful gene silencing experiments when using ibidi’s µ-Slides in live cell imaging. Both reagents combine low toxicity with outstanding transfection results and a simple, rapid protocol. Microscopic analysis of transfection experiments is an easy method for analyzing transfection efficiency, or for directly monitoring the effects and localization of synthesized proteins. ibidi’s µ-Slides VI 0.4 (see page 78) allow for cell culture, transfection, and microscopy to occur all in one slide. This makes them perfect for applications like imaging of fluorescent proteins, protein localization, or gene regulation studies.

Cell membrane

ENDOCYTOSIS

Transduction LifeAct protein TRANSLATION

Endosome

mRNA RELEASE OF DNA

TRANSCRIPTION

Transduction of eukaryotic cells using an adenoviral vector, here rAV-LifeAct (see p. 33)

Method Comparison

40

The most common transfer method is the transfection of DNA (e.g., plasmids) or RNA (e.g., siRNA) into eukaryotic cells. This method allows transient and stable transfection of easy-to-access cells, but may lead to variable transfection efficiencies, depending on the cell type.

Cells that are difficult to transfect with plasmid DNA can easily achieve an efficiency of up to 100 % with transduction, a virus-mediated DNA transfer. Adenoviral vectors have proven to be a very successful tool in many different human and rodent cell types. Transduction efficiencies of up to 100 % can easily be achieved. Moreover, this method also gives access to difficult-to-transfect cells, such as primary cells. With the rAV-LifeAct adenoviral vectors (see page 33), ibidi provides a perfect tool for visualizing F-actin in difficult-to-access cells (e.g., endothelial cells) without interfering with the cytoskeletal dynamics.

Transfection

Adenoviral Transduction

Transient and stable protein expression

Only transient protein expression

Easy-to-transfect cells

Difficult-to-transfect cells

Variable transfection efficiency

Up to 100 % transduction efficiency

Biosafety level 1

Biosafety level 2


Membrane fusion is a novel and highly superior method to incorporate various molecules and particles into eukaryotic cells, and a strong strategy for functional studies and therapeutic approaches.

Specific liposomal carriers are able to attach and instantly fuse with plasma membranes in a physicochemicaldriven manner. ibidi’s new Fuse-It reagents efficiently use this mechanism and fuse with mammalian cell surfaces immediately upon contact. Therefore, this novel technique makes the transfer of molecules independent of biological processes, such as endocytosis, pinocytosis, or specific receptor binding.

Molecules

Liposomal Carrier

Membrane Fusion

Cytoplasm Nucleus

Membrane fusion makes it possible to effectively incorporate different classes of molecules into the cellular membrane or into the cytoplasm. Systematic adaptations of the liposome composition to the general characteristics of a defined molecular class guarantee fusion efficiencies up to 80 - 100% within only seconds to a few minutes. Additionally, the very short incubation times provide an almost unaffected cell behavior after the molecule transfer.

Cell Preparation

The incorporation of small liposomal carriers into the plasma membrane of mammalian cells is the idea behind all of ibidi’s Fuse-It products. ibidi offers specifically optimized products based on the molecular classes required for your research. Please find detailed information on the pages 42 - 46.

Fuse-It

Transfection, Transduction, Membrane Fusion

Membrane Fusion

CHO cells surface functionalized with biotin using Fuse-It-B (red) and subsequent binding of avidin-alexa488 (green)

Method Comparison Transfection and Fusion define two highly synergistic techniques vital for cell manipulation. Technical characteristics given below can help to provide a basis for experimental design. Transfection

Membrane Fusion

Transfer of nucleic acids

Transfer of lipids, proteins, particles, membrane components, functionalization molecules, etc.

Incorporation of liposomes into cells by endocytosis

Transfer of liposomes into cells by plasma membrane fusion

Transient and stable expression

Transient incorporation with natural, molecule-specific lifetimes that are in the range of hours to days

Procedure time: minutes to several hours

Procedure time: minutes

12 - 24 h of idle time before analysis

Direct analysis after incorporation

Typically high rates of protein expression

Adjustable, intermediate molecule concentrations

Transfection efficiency strongly varies between different cell types

High incorporation efficiency, largely independent of cell type

Biosafety level 1

No biosafety restrictions to biosafety level 1

41


in cooperation with

Fuse-It

Live Cell Imaging

beniag

Membrane fusion used as a novel and highly superior method of transiently incorporating various molecules and particles into eukaryotic cells

biotin

dye

lipid

membrane protein

soluble proteins

beads

Membrane Fusion

Liposomal Carrier

Cytoplasm Nucleus

0.5 min

3 min

5 min

• 80-100 % efficiency independent of cell type, cell density, and developmental state • Fast results within 1-30 minutes • Applicable for dyes, proteins, lipids, beads, etc. • Equally efficient for suspension cells, adherent cells, and even thick tissue layers • Fully biocompatible reagents • Just one product per molecule class The incorporation of small liposomal carriers into the plasma membrane of mammalian cells is the idea behind all of ibidi’s Fuse-It products. Liposomal carriers are able to attach and instantly fuse with plasma membranes in a physicochemical-driven manner. ibidi’s new Fuse-It reagents efficiently use this mechanism and fuse with mammalian cell surfaces immediately upon contact. Therefore, this novel technique makes the transfer of molecules independent of biological processes, such as endocytosis, pinocytosis, or specific receptor binding.

dye

Fuse-It-Color N

A fusion reagent used to efficiently color-label plasma membranes of living cells

EW

• Fusogenic liposomes are used as a carrier for lipophilic dyes to label cellular plasma membranes • Fusion process completed within 1 - 10 minutes • Various emission spectra available

Applications

CHO cells fused with Fuse-It green (top, left), Fuse-It red (top, right), Fuse-It dred (bottom, left) and Fuse-It IR (bottom, right) for 1 min. Specifications :

42

• Live cell labeling for various microscopic purposes and techniques • Labeling of single cell types for co-culture experiments • Labeling of cells for flow cytometry and FACS After fusion, cells can immediately be analyzed. Cell labeling by fusion is extremely efficient, and leads to sufficient labeling densities for most cell types already within seconds. Ordering Information:

Concentration

3 mM

Storage

-20 °C

Cat. No.

Description

Shelf life

6 months

60200 / 60201

Fuse-It green, ready to use, 100 µl / 400 µl solution, 3 mM, green fluorescent

ExMax /EmMax

484/501 nm (green) 549/565 nm (red) 644/665 nm (dark red) 750/780 nm (IR)

60202 / 60203

Fuse-It red, ready to use, 100 µl / 400 µl solution, 3 mM, red fluorescent

1

60204 / 60205

Fuse-It dred, ready to use, 100 µl / 400 µl solution, 3 mM, dark red fluorescent

1

60206 / 60207

Fuse-It IR, ready to use, 100 µl / 400 µl solution, 3 mM, infrared fluorescent

1

Pcs. / Box 1


A fusion reagent used to transfer water soluble proteins into the cytoplasm of living cells

N

EW

soluble proteins

• The lumen of fusogenic liposomes is used as a carrier for water soluble proteins • Fusion process completed within 1 - 20 minutes • Optimized for the transfer of low and intermediate protein amounts to prevent concentration-induced artifacts in cell behavior

Applications

1 min

• Transfer of labeled proteins or peptides into living cells for functional imaging, speckle analysis, FRAP, or single molecule analysis, etc. • Incorporation of antibodies or blocking antibodies into living cells • Blocking, induction, or replacement of protein induced/regulated signal cascades

Transferred proteins are instantly active inside the cells, and after fusion, cells can immediately be used for further analysis. Since proteins are freed directly into the cytoplasm, no partial or complete lysosomal degradation can occur, as is typical for other endosomal uptake-depending proteofection methods. Depending on your experimental needs, the fusion process can be monitored by fluorescence microscopy.

Transfection, Transduction, Membrane Fusion

Fuse-It-P

3 min

5 min Fuse-It-P vesicles were filled with LifeAct and fused with myofibroblasts. A bright staining of the actin cytoskeleton can be observed after 5 minutes.

NOTE Transfer of heavily charged proteins can affect fusion efficiency.

Ordering Information:

Specifications :

Cat. No.

Description

60220

Fuse-It-P, lyophilized, for 100 µl solution, 3 mM, infrared (IR) fluorescent

Concentration

3 mM

1

Storage

-20 °C 6 months 750/780 nm

Pcs. / Box

60221

Fuse-It-P, lyophilized, for 4 x 25 µl solution, 3 mM, IR fluorescent

1

Shelf life

60222

Fuse-It-P, lyophilized, for 400 µl solution, 3 mM, IR fluorescent

1

ExMax /EmMax

60223

Fuse-It-P, lyophilized, for 4 x 100 µl solution, 3 mM, IR fluorescent

1

43


Fuse-It-Beads N

Live Cell Imaging

EW

beads

A fusion reagent used to transfer beads and particles into the cytoplasm of living cells

• Efficient incorporation of beads and particles with diameters in the nm to µm range • Fusion process completed within 1 - 20 minutes

Applications

CHO cells fused with Fuse-ItBeads for 2 minutes with M-270 magnetic Dynabeads. Note: The transfer of positively charged beads can affect fusion efficiency.

• Magnetic µm-bead incorporation for magnetic tweezers experiments • Cell purification assays using paramagnetic nm-beads • Incorporation of antibody-coupled particles for labeling, speckle microscopy, electron microscopy, etc. After fusion, the cells can immediately be used for further analysis. Multiple bead or single bead transfer is possible, depending on incubation time and bead concentration. Depending on your experimental needs, the fusion process can be monitored by fluorescence microscopy.

Specifications : Ordering Information:

Concentration

3 mM

Storage

-20 °C

Cat. No.

Description

Shelf life

6 months

60420

Fuse-It-Beads, ready to use, 100 µl solution, 3 mM, IR fluorescent

1

ExMax /EmMax

750/780 nm

60421

Fuse-It-Beads, ready to use, 400 µl solution, 3 mM, IR fluorescent

1

Pcs. / Box

biotin

Fuse-It-B N

A fusion reagent used to biotinylate the surfaces of living cells

EW • Fusogenic liposomes are used as a carrier for the transfer of biotinylated lipids into the cellular plasma membrane • Fusion process completed within 1 - 20 minutes

Applications • Cell surface functionalization and targeting using biotin-avidin coupling • Cell separation using avidin-coated magnetic beads • Attachment of living cells to distinct precoated surfaces

44

CHO cells fused with Fuse-It-B (red) and subsequent binding of avidin-alexa488 (green)

After fusion, the cells can immediately be used for further analysis. Biotin can be visualized by anti-biotin antibodies or bound to avidin, neutravidin, and streptavidin, respectively. Depending on your experimental needs, the fusion process can be monitored by fluorescence microscopy.

Specifications :

Ordering Information:

Concentration

3 mM

Cat. No.

Description

Storage

-20 °C

60320

Fuse-It-B, ready to use, 100 µl solution, 3 mM, green fluorescent

Pcs. / Box 1

Shelf life

6 months

60321

Fuse-It-B, ready to use, 400 µl solution, 3 mM, green fluorescent

1

ExMax /EmMax

484/501 nm (green); 750/780 nm (IR)

60322

Fuse-It-B, ready to use, 100 µl solution, 3 mM, IR fluorescent

1

60323

Fuse-It-B, ready to use, 400 µl solution, 3 mM, IR fluorescent

1


Fuse-It-L

N

EW

A fusion reagent used to incorporate lipids or amphipathic molecules into the plasma membrane of living cells

• Fusogenic liposomes used as a lipid carrier • Fusion process completed within 1 - 10 minutes

Applications • Lipid imaging for monitoring turnover, traffic, and functional analysis in living cells • Lipid raft, lipid microdomain, and lipid diffusivity measurements • Blocking, induction, or replacement of lipid induced/regulated signal cascades • Re-incorporation of lipids into mutant cells affected in lipid biosynthesis After fusion, cells can immediately be used for further analysis. If necessary, more than one lipid of interest can be incorporated at once. Depending on your experimental needs, the fusion process can be monitored by fluorescence microscopy.

Bodipy FL C12-galactocerebroside and TopFluor cholesterol were incorporated into the plasma membrane of CHO cells using Fuse-It-L (5 min incubation). Specifications :

Ordering Information: Cat. No.

Description

Concentration

3 mM

60210

Fuse-It-L, lyophilized, for 100 µl solution, 3 mM, IR fluorescent

1

Storage

-20 °C

60211

Fuse-It-L, lyophilized, for 4 x 25 µl solution, 3 mM, IR fluorescent

1

Shelf life

6 months

60212

Fuse-It-L, lyophilized, for 400 µl solution, 3 mM, IR fluorescent

1

60213

Fuse-It-L, lyophilized, for 4 x 100 µl, solution, 3 mM, IR fluorescent

1

ExMax /EmMax

750/780 nm

Pcs. / Box

Transfection, Transduction, Membrane Fusion

lipid

Fuse-It-T N

EW

A fusion reagent used to efficiently label cellular plasma membranes in tissue material or thick tissue sheets

• Fusogenic liposomes are used as a carrier for lipophilic dye • Fusion process completed within 10 - 30 minutes • Labeling of up to 10 stacked cell layers

Applications • Live cell imaging in a tissue environment for any kind of microscopic technique, including those optimized for thick specimens (e.g., confocal, two-photon, and light-sheet microscopy) • Labeling of tissue samples before microtome dissection After fusion, cells can immediately be analyzed. Accurate characterization of cell number, localization, and intercellular crosstalk is vital for an overall understanding of cell behavior in tissues. For the very first time, Fuse-It-T enables you to do all of these processes in just one single step. Upon request, additional labeling can be provided.

Rat embryonal rat heart sample labeled with Fuse-It-T for 10 minutes and imaged as Z-stack Specifications :

Ordering Information: Pcs. / Box

Concentration

3 mM

Storage

-20 °C

Cat. No.

Description

60260

Fuse-It-T, ready to use, 100 µl solution, 3 mM, red fluorescent

1

Shelf life

6 months

60261

Fuse-It-T, ready to use, 400 µl solution, 3 mM, red fluorescent

1

ExMax /EmMax

549/565 nm

45


membrane protein

Fuse-It-MP N

A fusion reagent used to incorporate transmembrane or membraneassociated proteins into the plasma membrane of living cells

Live Cell Imaging

EW

• Transfer protein while maintaining the natural membrane composition • Detergent-dependent protein purification is no longer necessary • Fusion process completed within 1 - 20 minutes

Applications • Blocking, induction, or replacement of membrane protein induced/regulated signal cascades • Re-incorporation of membrane proteins into mutant cells • Provides cells with new functions • Imaging for functional analysis of membrane protein turnover and traffic in living cells

Transmembrane proteins can be incorporated into liposomes, while still maintaining the natural membrane composition. This outstanding technique continuously preserves the natural lipid environment for all types of membrane proteins, and therefore prevents degradation. Also, transferred proteins are instantly active inside the cells, and after fusion, the cells can immediately be used for further analysis. Depending on your experimental needs, the fusion process can be monitored by fluorescence microscopy.

Bacteriorhodopsin transmembrane protein (red) was incorporated into CHO cells using Fuse-It-MP liposomes. Fusion efficiency is given in green. Yellow = overlay

NOTE Transfer of heavily charged or highly glycosylated proteins can affect fusion efficiency.

Specifications :

46

Ordering Information:

Concentration

3 mM

Cat. No.

Description

Storage

-20 °C

60250

Fuse-It-MP, ready to use, 100 µl solution, 3 mM, IR fluorescent

1

Shelf life

6 months

60251

Fuse-It-MP, ready to use, 400 µl solution, 3 mM, IR fluorescent

1

ExMax /EmMax

750/780 nm

Pcs. / Box


N

EW

A support reagent for enhancing the efficiency of viral transfer into difficult-to-transduce mammalian and rodent cells

• 20- to 50-fold enhanced adenovirus transduction • A non-toxic, peptide-based solution • Gives access to nearly all cell types

Applications • Adenovirus transduction

Technical Features: • Gives access to cells that express little CAR (Coxsackie Adenovirus Receptor) Adenovirus transduction can be challenging especially when cells express little CAR (Coxsackie Adenovirus Receptor), which is the key to infecting cells. The ibiBoost™ Adeno Adenovirus Transduction Enhancer has been specifically developed to improve the uptake of adenovirus vectors (e.g., rAV-LifeAct Adenoviral Vectors; see page 33) into cells and cell lines that are normally difficult to transduce. ibiBoost reagent is unique, because it is based on an adenovirus binding peptide. Due to this factor, ibiBoost changes the adenovirus surface in a way that the uptake of the adenovirus is improved for all permissive cell types, and it allows transduction of cell types previously not accessible to adenovirus transduction. Unlike chemical transduction enhancers, ibiBoost has no toxic effects. The use of ibiBoost is especially recommended if high ”Multiplicities of Infection“ (MOI), exceeding 500, have to be applied to a particular cell type.

ibiBoost

Transfection, Transduction, Membrane Fusion

ibiBoost™ Adeno Adenovirus Transduction Enhancer

CAR

ibiBoost Adeno bridges the adenovirus surface to the cell membrane, thereby enhancing the gene expression by 20- to 50-fold into cell types that express little of the primary adenovirus receptor CAR.

ibidi APPLICATION NOTE In ibidi’s application note Adenoviral Transduction of Human Cells (AN28)

ibiBoost Adeno significantly enhanced GFP expression in rodent NIH-3T3 cells.

Ordering Information: Cat. No.

Description

50301

ibiBoost Adeno Adenovirus Transduction Enhancer Solution, 50 transductions

you will find a detailed protocol for handling recombinant adenoviruses, and designing an approach for transducing human cells.

Specifications : Pcs. / Box

Volume

125 μl

1

Amount

For 50 transductions à 1 × 107 IU

Storage

4°C

NOTE: Please note that you will be working with samples containing an infectious virus. Follow the recommended NIH guidelines for all materials containing BSL–2 organisms.

47


ibiClone™ Adeno Adenovirus Cloning Kit N

A kit for recombination-based, adenoviral vector generation in E.coli, using BAC recombination technology

Live Cell Imaging

EW

• Extremely fast vector cloning and production in 2 weeks • Reliable results due to the sophisticated BAC selection system • Highest cloning efficiency available using BAC technology (100% stable, positive clones) • Simple and ready-to-use kit

Applications • Gene (over)expression in mammalian cells • Gene knockdown in mammalian cells

Technical Features: • A patent-pending adenovirus vector technology • 100% stable clones created through BAC technology Schematic Overview of the ibiClone™ Adeno Cloning System Plasmid including gene of interest (GOI)

1. Cloning

pCMV O6A5-GOI

2. Transformation 3. Recombination

BAC

recombined BAC with GOI

4. Linearization

Virus genome with GOI

5. Virus reconstitution in HEK293 cells

Specifications : Kit pO6A5-CMV schuttle Components vector E.coli BA5-FRT electrocompetent cells SOC medium

48

Amount

10 reactions

Storage

Bacteria: -80 °C Plasmid: -20 °C SOC medium: Room temperature

• 100% positive clones created through BAC technology The ibiClone™ Adeno Cloning System is a novel, innovative technology that allows for the fast and convenient generation of recombinant adenoviruses, in as little as two weeks. The system is basically composed of just two major elements: Component 1: A small shuttle vector (pO6A5), in which the transgene is cloned. Component 2: Electrocompetent BA5-FRT E. coli cells with a BAC vector that confers resistance to chloramphenicol. Contained on this BAC vector is an Ad5 genome that is devoid of the left ITR, the packaging signal, the E1 sequences, and 720 bp of the E3 region. Following the transformation of the shuttle vector into the electrocompetent BA5-FRT E.coli cells, an Flp recombinase-mediated recombination occurs between the shuttle and the BAC vector. Due to a highly sophisticated selection system, only cells containing a recombined BAC vector can grow and form bacterial colonies. As a result, almost 100% of the screened colonies contain BAC vectors with the correctly recombined transgene. Because of this factor, the ibiClone™ Adeno Cloning System requires very limited ‘hands on’ time, from the shuttle vector transformation to the isolation of virus particles. Virus yields and quality are absolutely comparable to those obtained with established adenoviral cloning systems.

Ordering Information: Cat. No.

Description

60661

ibiClone Adeno Adenovirus Cloning Kit: pO6A5 shuttle vector for gene overexpression (CMV promoter), electrocompetent BA5 FRT cells, 10 reactions

Pcs. / Box 1

60662

ibiClone Adeno Adenovirus Cloning Kit: pO6A5 shuttle vector for silencing (U6 promoter), electrocompetent BA5 FRT cells, 10 reactions

1

60663

Consumables ibiClone Adeno Adenovirus Cloning Kit: electrocompetent BA5 FRT cells, 10 reactions

1

NOTE: Please note that you will be working with samples containing an infectious virus. Follow the recommended NIH guidelines for all materials containing BSL–2 organisms.


N

EW

A kit for chromatography-based adenovirus purification from a cell pellet

• Fast preparation in only 20 minutes • Just 4, easy-to-perform steps • Efficient purification with significantly high yields (109 – 1010 of infectious units (IU) from a single 15 cm dish)

Applications • Adenovirus purification, after amplification in HEK293 cells

Technical Features: Significantly Higher Virus Yields, Compared to the Industry Standard

• Spin-column-based adenovirus purification, directly from cell pellet • Anion exchange membrane with extraordinarily high binding capacity for adenoviruses

The ibiPure™ Adeno Adenovirus Purification Kit is a robust and reliable, spin-column-based system for quick and convenient adenoviral purifications, showing high recovery rates. The technology is based on an anion exchange membrane with extraordinarily high binding capacity for adenoviruses. The purification protocol is almost as simple as that of most plasmid purification systems. Just equilibrate your column, load your sample, wash, and then elute your purified virus.

2,0*1010 Total yield [IU]

Cellular debris and medium-derived proteins in non-purified adenovirus preparations often cause toxic effects and harm viral activity during storage. Hence, the purity of adenovirus preparations is critical for success.

Transfection, Transduction, Membrane Fusion

ibiPure™ Adeno Adenovirus Purification Kit

1,5*1010 1,0*1010 0,5*1010 A

B

C

D

E

Adenovirus preparation ibiPureTM Adeno Industry standard

Harvest and lyse cells Pellet cell debris Glycerol

Infection of HEK293 cells

Equilibrate column

Ordering Information: Cat. No. Description

Load sample

Wash column

Elute purified virus

Specifications : Pcs. / Box

60681

ibiPure Adeno Adenovirus Purification Kit, 12 preps

1

60682

ibiPure Adeno Adenovirus Purification Kit, 24 preps

1

NOTE: Please note that you will be working with samples containing an infectious virus. Follow the recommended NIH guidelines for all materials containing BSL–2 organisms.

Kit Components

Equilibration Buffer 1-3, Loading Buffer, Wash Buffer, Elution Buffer, Glycerol, Spin Columns, Collection Tubes

Expected Viral Titer

10 9 - 5x1010 IU/ml*

Final Volume of Purified Adenovirus

500 µl

Viral Serotype

The kit is designed for the purification of adenovirus serotype 5. The user must test all other serotypes.

Storage

Room temperature

* Titer was determined using the immunocytochemical detection of adenovirus hexon protein in infected cells.

49


Torpedo DNA Transfection Reagent

Live Cell Imaging

A reagent for transfection of eukaryotic cells with plasmid DNA; optimized for use with ibidi’s µ-Slides and µ-Dishes

• Superb biocompatibility with low cytotoxicity for use in live cell imaging • Transfection efficiency optimized for microscopic assays

Applications • Fluorescence microscopy, live cell imaging, and confocal fluorescence microscopy • Imaging of fluorescent proteins after transient or stable plasmid DNA transfection • Protein localization with fluorescence labels

HT-1080 cells were transfected with pCMV LifeAct-TagGFP2 (see page 33) using Torpedo DNA . The cells were imaged 24 hours afterwards showing high transfection efficiency.

Lipid based transfection reagents often interact with plastic surfaces and therefore affect transfection efficiency and cell viability. ibidi’s Torpedo DNA reagent has been specifically optimized for use with ibidi’s plastic materials. This results in a reagent with very low cytotoxicity as well as high transfection efficiency. Transfection Protocol for µ-Slide 8 well (see page 66):

12 9

3 6

I. Plasmid

15-20 min

Medium or PBS

ibidi MOVIE View a detailed transfection protocol for adherent cells in an ibidi microscopy chamber:

II. Torpedo DNA Transfection Reagent

Cell Transfection Using Torpedo DNA (MV 24) on www.ibidi.com.

Specifications :

50

Ordering Information:

Composition

Proprietary cationic lipids

Cat. No.

Description

Assays

Up to 500 (24-well) or up to 150 (6-well) with 0.5 ml reagent

60610

Torpedo , Transfection reagent for eukaryotic cells using plasmid DNA and RNA, ready to use, 0.5 ml

1

Storage

- 20 °C

60611

Torpedo DNA , Transfection reagent for eukaryotic cells using plasmid DNA and RNA, ready to use, 1.5 ml

1

60612

Torpedo DNA , Transfection reagent for eukaryotic cells using plasmid DNA and RNA, ready to use, 2 x 2.0 ml

1

Pcs. / Box

DNA


N

EW

A reagent for transfection of mammalian cells with siRNA or miRNA; optimized for use with ibidi’s µ-Slides and µ-Dishes

• High transfection efficiency • Superb biocompatibility with low cytotoxicity for use in live cell imaging

48 h

siRNA control

48 h

siRNA EGFP

• Compatible with various cell lines

Applications • Gene silencing experiments • RNA research, e.g., epigenetics • Protein interaction studies

Technical Features: • ≥ 90% silencing efficiency for transient knockdown of mammalian gene expression

Cells – stably expressing EGFP – were seeded on a µ-Plate 24 well (see page 89) and transfected with siRNA against EGFP or non-specific, control siRNA using Torpedo siRNA . They were imaged after 24, 48, and 72 hours showing a knockdown of EGFP expression.

• Low siRNA concentration (1 nM) sufficient • Reagent compatible with serum Torpedo siRNA Transfection Reagent is specially designed for the transfection of small RNA (siRNA & miRNA) into mammalian cells. Lipid based transfection reagents often interact with plastic surfaces and therefore affect transfection efficiency and cell viability. ibidi’s Torpedo siRNA reagent has been specifically optimized for use with ibidi’s plastic materials. This results in a reagent with very low cytotoxicity as well as high transfection efficiencies.

Transfection, Transduction, Membrane Fusion

Torpedo siRNA Transfection Reagent

Reverse siRNA Transfection Protocol for µ-Plate 24 well (see page 89):

I. Step

II. Step Mix gently by pipetting

12 9

Incubate without further mixing 24 - 72 hours prior to analysis

3 6

Incubate 15 mins at RT Prepare 1x Buffer T in sterile water

1. 1x Buffer T 2. Torpedo siRNA 3. RNA

Add cell suspension to wells

Prepare cell suspension in complete medium

Ordering Information:

Specifications :

Cat. No.

Description

Pcs. / Box

60620

Torpedo , Transfection reagent for mammalian cells using siRNA and miRNA, ready to use, 0.5 ml

1

60621

, Transfection reagent for mammalian cells using siRNA Torpedo and miRNA, ready to use, 1.5 ml

1

60622

Torpedo siRNA , Transfection reagent for mammalian cells using siRNA and miRNA, ready to use, 2 x 2.0 ml

1

siRNA

siRNA

Composition

Proprietary lipids in water

Assays

Up to 1500 (96-well), or up to 375 (24-well) with 1.5 ml reagent

Storage

- 20 °C

51


Collagen Type I N

A high quality, rat tail collagen solution used for creating 3D collagen gels (e.g., in chemotaxis assays)

Live Cell Imaging

EW

• Provides in vivo–like ECM (extracellular matrix) structures • High concentrated collagen solution – usable for a variety of gel concentrations • Fast polymerizing for optimal cell distribution in gel • Non-pepsinized, native collagen for a close-to-nature situation

Applications • Chemotaxis assays in firm 3D collagen gels • Leukocyte and tumor cell migration • 3D cell microscopy • Culturing cells on thin or thick collagen layers • Coating of cell cultureware to promote cell attachment • Differentiation studies – 2D versus 3D High resolution microscopy during chemotaxis and cell polarization, using the HT-1080 LifeAct cell line (see page 34)

• Highly suited for live cell imaging and fluorescence microscopy

Technical Features: • High-quality purified protein from rat tendons • Ideal for use in combination with µ-Slide Chemotaxis 3D (see p. 109) • Ready to use • Highly standardized manufacturing process • Stringent quality standards Cell spheroid (or micro tumor) on top of, or embedded in, a 3D matrix for long-term cultivation. The upper well provides nutrients by means of diffusion. Specifications : Rat tail tendon

Appearance

Optically clear viscous liquid

Extraction

Acid, non-pepsinized

Purity

> 90 % by SDS PAGE

Sterility

Sterile, for cell culture

Contaminants

Negative for DNA, bacteria, fungi, and mycoplasma

Growth factors None

52

Supplied in 17.5 mM acetic acid (0.1%)

pH

pH 3.8

Functional control

3D gelling and 2D coating test in cell culture

Storage

• Storage at -20°C for well-defined quality and reproducibility

ibidi APPLICATION NOTE

Source

Formulation

• Simple and easy protocol and handling

-20°C

The Application Note Collagen I Gel in 3D Cell Culture (AN 26) provides detailed protocols on how to prepare collagen I gels with different cell culture media and different types of collagen.

Ordering Information: Cat. No.

Description

50201

Collagen Type I, rat tail, non-pepsinized, 5 mg / ml, 1 x 5 ml

Pcs. / Box 1

50202

Collagen Type I, rat tail, non-pepsinized, 5 mg / ml, 4 x 5 ml

1

50203

Collagen Type I, rat tail, non-pepsinized, 5 mg / ml, 1 x 100 ml

1

50204

Collagen Type I, rat tail, non-pepsinized, 10 mg / ml, 1 x 5 ml

1

50205

Collagen Type I, rat tail, non-pepsinized, 10 mg / ml, 4 x 5 ml

1

50206

Collagen Type I, rat tail, non-pepsinized, 10 mg / ml, 1 x 100 ml

1


ibidi Anti-Evaporation Oil

Cell Preparation & Reagents

A biocompatible silicone oil used to prevent medium evaporation in cell culture applications

• Excellent reduction of medium evaporation • Ready to use • Compatible with all ibidi products and cell culture applications

Applications • Reducing evaporation in channel slides or open wells • Sealing low volumes of culture medium • Long-term microscopy assays

Technical Features • Specially designed for all ibidi µ-Slides, µ-Dishes, and µ-Plates • Non-drying and volume stable • Dry-heat sterilized for direct use • Highly gas permeable, thus providing excellent cell cultivation conditions • Chemically stable • Viscosity optimized for cell culture • Compatible with microscopy

Channel Slide

Open Well Format

ibidi APPLICATION NOTE

Fill channel µ-Slide or µ-Dish with cells and medium as usual

ibidi’s Application Note Avoiding Evaporation (AN 12) recommends various techniques to control and decrease evaporation in cell culture.

Overlay the medium’s surface with ibidi Anti-Evaporation Oil

Ordering Information:

Specifications:

Cat. No.

Description

50051

ibidi Anti-Evaporation Oil, silicone oil for cell culture, dry-heat sterilized, 125 ml

Pcs. / Box 1

Appearance

Optically clear in dry atmosphere

Density (20 °C)

1.05 g / ml

Viscosity (20 °C)

Ca. 200 mPa . s

Refractive index nD

1.453

Temperature stability

121 °C (dry-heat only)

Storage of product

Dark, room temperature

53


ibidi Freezing Medium Classic | Direct | HRM N

Cell freezing media for standard cell lines, hybridoma cell lines, or ES / iPS cells

Live Cell Imaging

EW

• No preliminary or sequential freezing required • Extremely high recovery rates • Very competitive pricing

Technical Features • Direct freezing at -80 °C, or by using liquid nitrogen • Tested for sterility, endotoxins, mycoplasma, fungi, and bacteria • Specialized formulation for various applications

Two different cell lines before freezing and 12 months after. The cells were stored at -80°C

Before freezing

ibidi Freezing Medium Direct

ibidi Freezing Medium Classic

Very Effective Cryoprotection Shown in Selected Cell Tests: P3U1 (mouse myeloma cell line)

K526 (human leukemia cell line)

OKT4 (mouse hybridoma)

Human gastric epithelial cells

ibidi Freezing Medium Classic

95 %

73 %

100 %

100 %

Serum Medium

95 %

70 %

100 %

62 %

Serum-free Medium

70 %

60 %

92 %

56 %

Storage period

1 year

1 year

1 year

10 months

Survival rate

Specifications: ibidi Freezing Medium

Classic

Direct

HRM

Application Area

Standard cell lines

Standard cell lines + Hybridoma Cell Lines

Especially primate ES and iPS cells

High Throughput

No

Yes

No

Serum

Bovine Serum Albumin

Bovine Serum Albumin

Human Serum Albumin

Appearance

Optically clear

Optically clear

Optically clear

Storage of product

4 °C

4 °C

4 °C

Ordering Information:

54

Cat. No.

Description

80021

ibidi Freezing Medium Classic, with Bovine Serum Albumine, sterile, 10 ml

Pcs. / Box 1

80022

ibidi Freezing Medium Classic, with Bovine Serum Albumine, sterile, 5 x 20 ml

1

80023

ibidi Freezing Medium Classic, with Bovine Serum Albumine, sterile, 120 ml

1

80024

ibidi Freezing Medium Direct (no cell centrifugation required), with Bovine Serum Albumine, sterile, 20 ml

1

80025

ibidi Freezing Medium HRM (for ES and iPS cells), with Human Serum Albumine, no animal components, sterile, 20 ml

1


ibidi Mounting Medium

Cell Preparation & Reagents

A mounting medium that is optimized for fluorescence microscopy and ibidi’s µ-Slides and µ-Dishes

• Ready to use in widefield fluorescence, confocal microscopy, and immunofluorescence assays • No autofluorescence – bright fluorescence signals • Dropper bottle for exact dispensing into channels or open wells

Technical Features • Storage of stained slides and dishes - for days or for weeks • Specially designed for all ibidi µ-Slides, µ-Dishes, and µ-Plates • Contains antioxidant agent to preserve fluorochromes • Optimal pH for fluorescence signals • Non-drying, non-hardening, and volume stable

Specifications: Appearance

Optically clear

pH

8.5 - 9.0

Refractive index nD

1.42 - 1.44

Storage of product

Dark, room temperature

Storage of mounted Dark, 4 °C samples

Easy well filling (e.g., µ-Dish)

Easy channel filling (e.g., µ-Slide VI)

Ordering Information: Cat. No. Description 50001

Pcs. / Box

ibidi Mounting Medium, for fluorescence microscopy, 15 ml

1

N

An immersion oil that is fully compatible with all µ-Slides, µ-Dishes, and µ-Plates

EW

ibidi Immersion Oil

• Provides excellent imaging quality • Compatible with all ibidi products • No autofluorescence – bright fluorescence signals

Specifications: Appearance

Optically clear

Refractive index nD

1.515

Background

Ultra-low fluorescence background

Storage of product

Dark, room temperature

Ordering Information: Cat. No.

Description

50101

ibidi Immersion Oil, for fluorescence microscopy, 15 ml

Pcs. / Box 1

55


Live Cell Imaging

Customer Feedback

„We used the Torpedo siRNA Transfection Reagent for the transfection of human retinal pigment epithelial cells and experienced a reproducible and efficient knock-down of our protein of interest; even with really low amounts of siRNA.“ Dr. Juliane Merl Research Unit Protein Science Helmholtz Zentrum München Germany

“I am also using the ibidi Gas Incubation and Temperature Control Systems, which are robust and guarantee perfect stability. Very importantly, the product developers at ibidi provide a great support for the implementation of their systems. Thanks to ibidi for developing cell imaging products of such great quality!” Loïc Dupré PhD INSERM UMR 1043 Purpan University Hospital Toulouse France

“We use LifeAct-TagRFP and LifeAct-TagGFP2 plasmids in primary human macrophages. The LifeAct probes put us in the position to detect F-actin-rich adhesion structures, without the drawback of disturbing their dynamics. The signal is bright and clear without the background of nonintegrated G-actin. LifeAct gives us the opportunity to highlight F-actin-enriched, cytoskeletal organizations, without the disadvantages of fluorophore-tagged actin overexpression.” Dr. Christiane Wiesner University Medical Center Hamburg-Eppendorf Hamburg Germany

References Protein Kinase D1 Maintains the Epithelial Phenotype by Inducing a DNA-Bound, Inactive SNAI1 Transcriptional Repressor Complex L. I. Bastea, H. Döppler, B. Balogun and P. Storz PLoS ONE, 2012, 10.1371/journal. pone.0030459 Flotillin-involved uptake of silica nanoparticles and responses of an alveolar capillary barrier in vitro J. Kasper, M. I. Hermanns, C. Bantz, S. Utech, O. Koshkina, M. Maskos, C. Brochhausen, C. Pohl, S. Fuchs and R. E. Unger European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft für Pharmazeutische Verfahrenstechnik e.V., 2012, 10.1016/j.ejpb.2012.10.011

56

Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification A. Csiszár, N. Hersch, S. Dieluweit, R. Biehl, R. Merkel, B. Hoffmann Bioconjug Chem, 2010, 21:537-543 Fluorescent lipids: functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization C. Kleusch, N. Hersch, B. Hoffmann, R. Merkel, A. Csiszár Molecules, 2012, 17:1055-73. Arraying Cell Cultures Using PEG-DMA Micromolding in Standard Culture Dishes A.-K. Marel, S. Rappl, A. Piera Alberola and J. O. Rädler Macromolecular Bioscience, 2013, 10.1002/mabi.201200400

Arp2/3 Is Critical for Lamellipodia and Response to Extracellular Matrix Cues but Is Dispensable for Chemotaxis C. Wu, Sreeja B. Asokan, Matthew E. Berginski, Elizabeth M. Haynes, Norman E. Sharpless, Jack D. Griffith, Shawn M. Gomez and James E. Bear Cell, 2012 Decoding the Regulation of Mast Cell Exocytosis by Networks of Rab GTPases N. P. Azouz, T. Matsui, M. Fukuda and R. Sagi-Eisenberg The Journal of Immunology, 2012, 10.4049/jimmunol.1200542 Lasp-1 Regulates Podosome Function M. Stölting, C. Wiesner, V. van Vliet, E. Butt, H. Pavenstädt, S. Linder and J. Kremerskothen PLoS ONE, 2012, 10.1371/journal. pone.0035340


IMMUNOFLUORESCENCE (IF) . . . . . . . . . . . 74

µ-Dish Family . . . . . . . . . . . . . . . . . . . . . . . . . . 60

µ-Slide VI 0.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

µ-Dish 35mm, high . . . . . . . . . . . . . . . . . . . . . . . . . 61

sticky-Slide VI 0.4 . . . . . . . . . . . . . . . . . . . . . . . 79

µ-Dish 35mm, low . . . . . . . . . . . . . . . . . . . . . . . . . . 61

µ-Slide VI – flat . . . . . . . . . . . . . . . . . . . . . . . . 80

µ-Dish 35mm, high glass bottom . . . . . . . . . . . . . . 62

µ-Slide 18 well – flat . . . . . . . . . . . . . . . . . . . . 84

DIC Lid for µ-Dishes . . . . . . . . . . . . . . . . . . . . 62

12 well Chamber, removable . . . . . . . . . . . . . 85

µ-Dish

50mm, low

. . . . . . . . . . . . . . . . . . . . . . . . . 62

µ-Dish

35mm, high

ESS . . . . . . . . . . . . . . . . . . . . . 63

SCREENING . . . . . . . . . . . . . . . . . . . . . . . . . . 86

µ-Dish Grid-500 µ-Dish 35mm, high Grid-500 . . . . . . . . . . . . . . . . . . 64

µ-Plate Family . . . . . . . . . . . . . . . . . . . . . . . . . 88

35mm, low

µ-Plate 24 well . . . . . . . . . . . . . . . . . . . . . . . . . 89

µ-Dish 35mm, high glass bottom Grid-50 µ-Dish 35mm, high glass bottom Grid-500 . . . . . . . 65

µ-Plate 96 well . . . . . . . . . . . . . . . . . . . . . . . . . 89

µ-Slide 2 well | 4 well | 8 well . . . . . . . . . . . . . 66

µ-Plate 384 well . . . . . . . . . . . . . . . . . . . . . . . 89

µ-Slide 2 well

Ph+

| 4 well

Ph+

. . . . . . . . . . . . . . . 67

µ-Slide 2 well | 4 well | 8 well glass bottom µ-Slide 2 well Ph+ | 4 well Ph+ glass bottom . . . . 68 DIC Lid for µ-Slides . . . . . . . . . . . . . . . . . . . . . 68 sticky-Slide 8 well . . . . . . . . . . . . . . . . . . . . . . 69

Cell-Based Microscopy Assays

CELL-BASED MICROSCOPY ASSAYS . . . . . 58

µ-Slide 2 x 9 well . . . . . . . . . . . . . . . . . . . . . . . 70 µ-Slide I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 micro-Insert 4 well Family . . . . . . . . . . . . . . . . 72 Single micro-Insert 4 well in a 35 mm µ-Dish . . . . . . . . . . . . . . . . . . . . . . 73 micro-Insert 4 well 24 . . . . . . . . . . . . . . . . . . . 73 25 micro-Inserts 4 well for self-insertion . . . . . . . . . . . . . . . . . . . . . . . 73

57


Cell-Based Microscopy Assays

Cell-Based Microscopy Assays

Application examples using various ibidi µ-Slides and µ-Dishes

3D Cell Microscopy Numerous ibidi products are made for 3D applications. In 3D assays, cells are brought inside, or on top of, an in vivo-like gel matrix. As with all ibidi µ-Slides, cells are, in most cases, being analyzed using fluorescence microscopy. Cells in a 3D matrix with a chemical gradient for chemotaxis. Typical for lymphocytes, neutrophils, and T-cells. Use the µ-Slide Chemotaxis 3D (see page 109) Cell spheroid (or micro tumor) on top of, or embedded in, a 3D matrix for longterm cultivation. The upper well provides nutrients by means of diffusion. Use the µ-Slide Angiogenesis (see page 100) Cells in 3D matrix with applied interstitial flow in a channel Use the µ-Slide VI 0.4 (see page 131)

Co-Cultivation ibidi has developed two cell culture products for co-cultivation assays. When using the µ-Slide 2 x 9 well (see page 70), two cell types can be cultivated separately, while still sharing one liquid medium. Apart from wound healing assays, ibidi’s Culture-Inserts (see page 94) can also be used in several co-culture assay derivations. The µ-Slide 2 x 9 well allows for the cultivation of cell spheroids inside a gel matrix, in combination with feeder cells seeded in the outer wells. Cell Type 1

Cell Type 2

Culture-Inserts in a large Petri dish with various adherent cell types during cell seeding. After cell attachment, the different cell types grow separately from each other, sharing one medium.

58

Cell Type 1

Cell Type 2


Small Organisms

Stem Cell Differentiation With ibidi‘s specially designed walls in the micro-Insert 4 well (see page 72), stem cells can be followed during their differentiation. Cells cannot “hide” near sharp edges and the system is long-term stable for days and weeks of stem cell cultivation.

Side View

Top View Seeding

Sedimentation Differentiation

Specific Substrates

Cell-Based Cell Microscopy Preparation Assays

The small ibidi wells are also applicable for microscopy assays with small organisms or embryos (e.g., Xenopus eggs, Drosophila embryos, C. elegans, Danio rerio, etc.). For these assays, we recommend the 30 µl wells of the µ-Slide 18 well - flat (see page 84).

When a custom-specific bottom material is needed, the ibidi stickySlides, such as the sticky-Slide 8 well (see page 69) and the sticky-Slide I Luer (see page 129), provide a versatile platform. Coverslips with protein or DNA / siRNA treatments can be used, as well as different materials like plastic sheets or glass materials.

Micromanipulation Special ibidi µ-Dishes with low walls (i.e., µ-Dish 35 mm, low (see page 61), µ-Dish 50 mm, low (see page 62)) are perfectly suited for micromanipulation assays, such as intracellular injections, micropipette chemotaxis assays, or tissue treatment and microscopy.

Low Walls = Greater Access

59


µ-Dish Family

Cell-Based Microscopy Assays

Petri dishes with a coverslip-like bottom for high end microscopy

Lid with Lock Position Open position. Easy opening. Close position. For cell cultivation. Minimal evaporation. Lock position. For long-term studies. Almost no evaporation.* * Approx. 0.1 % per day (37 °C, humid), Lid sealed with silicone grease. Approx. 1 % per day (37° C, humid) Approx. 10 % per day (37° C, dry)

• Unique ibidi Standard Bottom combining

- Excellent cell culture conditions; and - Supreme optical quality for high resolution microscopy

• Ideal cell growth conditions provided by the ibiTreat surface • Lid with lock position, which minimizes evaporation • Suitable for DIC, when used with the special DIC lid

Technical Features • Standard format dish with a 35 mm or 50 mm diameter • Made of biocompatible material using no glues, consequently no cells are harmed • No autofluorescence • Compatible with all staining and fixation solvents • Rim for easy opening (except µ-Dish 35mm, low)

ibidi TIP Use the locking feature only if minimal evaporation is required, i.e., outside incubators, non humidified microscopy stages, etc.

ibidi Standard Bottom The special coverslip (ibidi Standard Bottom) provides superb optics. Furthermore, cell culture conditions have been extensively tested using this coverslip, by ibidi and numerous scientific users, with positive results in over 2,000 peer-reviewed publications.

ibidi Standard Bottom vs. ibidi Glass Bottom ibidi Standard Bottom

Optical properties Refractive index (nD 589 nm) Abbe number Thickness Material Autofluorescence Transmission Birefringence (DIC)

ibiTreat

Uncoated 1.52

ibidi Glass Bottom

Glass 1.52

56

55

#1.5 (180 µm)

#1.5H (170 mm)

Microscopy plastic

D 263M Schott borosilicate glass

Low

Low

Very high (even ultraviolet)

High (ultraviolet restrictions)

Low (DIC compatible*)

Low (DIC compatible*)

ibiTreat – tissue culture treated Uncoated – hydrophobic

Only pure glass

Possible

Possible

Other aspects Surface modifications Protein coatings Gas permeable

Yes

No

Material flexibility

High

Low

No

Yes

All kinds of fluorescence microscopy

TIRF and single photon

Breakable Recommended for

60

* Special glass DIC lids are available seperately (Cat. No. 80050, see page 62)


µ-Dish 35mm, high A 35 mm imaging dish with an ibidi Standard Bottom, used in high end microscopy and cell-based assays

• High walls with a standard height for easy handling • Also available with a relocation grid (see page 64)

Applications

2 ml 12 mm

• Immunofluorescence staining • Live cell imaging • Transfection

High Walls

Ordering Information:

Specifications :

Cat. No.

Description

81156

µ-Dish 35 mm, high, ibiTreat, tissue culture treated, sterile, high wall

81151

µ-Dish 35 mm, high, hydrophobic, uncoated, sterile, high wall

80050

DIC lid, with a glass insert, suitable for use with all ibidi 35 mm dishes, sterile

Ø µ-Dish

35 mm

60

Volume

2 ml

60

Growth area

3.5 cm2

Coating area using 400 µl

4.1 cm2

Ø observation area

21 mm

Height with / without lid

14 / 12 mm

Pcs. / Box

5

Microscopy

• Cell culture and high resolution fluorescence microscopy

Bottom: ibidi Standard Bottom

µ-Dish 35mm, low A 35 mm imaging dish with an ibidi Standard Bottom and low walls, used in high end microscopy and micromanipulation

• Large access for micromanipulation • Also available with a relocation grid (see page 64) 0.8 ml

Applications

7 mm

• Cell manipulation and microinjection • Fluorescence microscopy of both living and fixed cells

Low Walls

• Live cell imaging

Large access for micromanipulation

Ordering Information:

Specifications :

Cat. No.

Description

Ø µ-Dish

35 mm

80136

µ-Dish 35 mm, low, ibiTreat, tissue culture treated, sterile, low wall

60

Volume

800 µl

80131

µ-Dish 35 mm, low, hydrophobic, uncoated, sterile, low wall

60

Growth area

3.5 cm2

80050

DIC lid, with a glass insert, suitable for use with all ibidi 35 mm dishes, sterile

Coating area using 400 µl

4.1 cm2

Ø observation area

21 mm

Height with / without lid

9 / 7 mm

Pcs. / Box

5

Bottom: ibidi Standard Bottom

61


µ-Dish 35mm, high glass bottom

Cell-Based Microscopy Assays

A 35 mm imaging dish with a glass bottom, suitable for use in TIRF and single molecule applications

Polarized Light

• Perfect cell imaging thanks to the low thickness variability of the coverslips’ glass • Also available with a relocation grid (see page 65)

DIC beam path

Condenser Glas lid (ibidi Heating System)

Applications

DIC lid ibidi Standard Bottom or ibidi Glass Bottom

• Cultivation and microscopy of cell cultures • TIRF and single molecule applications

100x

Technical Features

Specifications :

• Bottom made from D 263 M Schott glass, 170 µm +/- 5 µm • May require coating to promote cell attachment

Ø µ-Dish

35 mm

Recommended volume

2 ml

Growth area

3.5 cm2

Ø observation area

21 mm

Height with / without lid

14/12 mm

Ordering Information:

Bottom: Glass coverslip No. 1.5H, selected quality, 170 µm +/- 5 µm

Cat. No.

Description

81158

µ-Dish 35 mm, high, glass bottom, D 263 M Schott glass, No. 1.5H selected quality, 170 µm +/- 5 µm, sterile

Pcs. / Box 60

DIC Lid for µ-Dishes • For use in Differential Interference Contrast (DIC) • Suitable for use with all 35 mm µ-Dishes Ordering Information: Cat. No.

Description

80050

DIC lid for µ-Dishes, with a glass insert, sterile

Pcs. / Box 5

µ-Dish 50mm, low A 50 mm imaging dish with an ibidi Standard Bottom and low walls, used for high end microscopy and micromanipulation • Largest growth area and volume of all ibidi products

3 ml

• Large access for micromanipulation

9 mm

Applications

Low Walls Large access for micromanipulation

• Cultivation of a maximum number of cells

Specifications :

62

• Cell culture and high resolution fluorescence microscopy • Micromanipulation of single cells

Ø µ-Dish

50 mm

Volume

3 ml

Growth area

7.0 cm

Coating area using 700 µl

7.9 cm2

Ø observation area

30 mm

Cat. No.

Description

Height with / without lid

12 / 9 mm

81136

µ-Dish 50 mm, low, ibiTreat, tissue culture treated, sterile

30

81131

µ-Dish 50 mm, low, hydrophobic, uncoated, sterile

30

Bottom: ibidi Standard Bottom

• Fluorescence microscopy of both living and fixed cells

2

Ordering Information: Pcs. / Box


µ-Dish 35mm, high ESS

N

EW

A 35 mm imaging dish with an Elastically Supported Surface (ESS) used for growing cells under “in vivo-near” conditions

• “in vivo-near” cell culture on elastically supported surface • High resolution microscopy on soft cell culture surface

s G la s

Pe tri di sh ib es id iS Bo ta tto nd m ard

ue

ue Ti ss

d

is s ft T

iu m

M

ed

So

• Live cell imaging of cell differentiation on soft surface

H ar

GPa MPa

Soft

ue

• Immunofluorescence stainings

Stiff

Ti ss

• Cellular mechanics research

kPa

• Cell culture under “in vivo-near” conditions, e.g. cardiomyocytes

1012 11 10 1010 9 10 108 107 106 105 104 103 102

• Transfection • Suitable for DIC, when used with a DIC lid (see page 62)

Microscopy

Applications

Surface elasticity [Pa]

• Based on the standard µ-Dish 35 mm, high (see page 61)

Cell medium

Technical Features

Coating

• 40 μm thick PDMS (Polydimethylsiloxane) layer on top of a 100 μm thick glass cover • Available stiffness: 1.5 kPa, 15 kPa, and 28 kPa

Elastic surface

0.04 mm

Glass support

0.1 mm

• Biocompatible soft cell culture material • High walls with a standard height for easy handling • Fully compatible with the ibidi Heating System (see page 15)

Differentiated mouse fibroblasts cultured on elastic surface (ESS 28 kPa) green: zyxin (Alexa 488 conjugated antibody) red: alpha- smooth muscle actin (Cy5 conjugated antibody) (courtesy R. Merkel, Jülich, Germany)

Ordering Information:

100 x

ibidi TIP For a detailed explanation on the ESS technology please refer to page 174.

Specifications :

Cat. No.

Description

81291

µ-Dish 35 mm, high ESS, uncoated elastic surface with a stiffness of 1.5 kPa, sterile, Ø 35 mm, 2 ml volume

Pcs. / Box 30

81391

µ-Dish 35 mm, high ESS, uncoated elastic surface with a stiffness of 15 kPa, sterile, Ø 35 mm, 2 ml volume

30

81191

µ-Dish 35 mm, high ESS, uncoated elastic surface with a stiffness of 28 kPa, sterile

30

81199

µ-Dish 35 mm, high ESS variety pack, uncoated elastic surface with a stiffness of 1.5, 15, and 28 kPa, sterile

3 x 10

Ø µ-Dish

35 mm

Volume

2 ml

Growth area

3.5 cm2

Coating area using 800 µl 4.1 cm2 Ø observation area

21 mm

Height with / without lid

14 / 12 mm

Bottom: Elastic surface (40 µm) on glass coverslip (100 µm)

63


µ-Dish 35mm, low Grid-500 µ-Dish 35mm, high Grid-500

Cell-Based Microscopy Assays

A 35 mm imaging dish with an ibidi Standard Bottom, low / high walls and an imprinted 500 µm relocation grid

• Ideal for relocating cells or cell clusters • Unique ibidi Standard Bottom combining

- Excellent cell culture conditions; and - Supreme optical quality for high resolution microscopy

Applications • Relocating cells or cell clusters, e.g., transfected cells for clone picking • Counting events per defined area (e.g., for calculating transfection efficiency)

Grid-500

• Providing a reference structure for cell movements • Treatment of distinct single cells, such as in microinjection

460 µm

• Following axon growth on a defined scale bar 500 µm

40 µm

Technical Features

< 5 µm

• Lettered and numbered 1 x 400 squares (A-U; 1-20) • Large access for micromanipulation in µ-Dish 35mm, low Grid-500

Layout Grid-500 i d i b i A B C D E F G H I

i d i b i A B

C

D E

F

G H I

K

A B

C

D E

F

G H I

K

L

M N O P

Q R

S

T

U

L

M N O P

Q R

S

T

U

1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 20

11 12 13 14 15 16 17 18 19 20 A B

C

D E

F

G H I

K

11 12 13 14 15 16 17 18 19 20 L

M N O P

Q R

S

T

K

L M N O P Q R S T U 1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10 A B C D E F G H I

K

1 2 3 4 5 6 7 8 9 10

500 µm

L M N O P Q R S T U

U

11 12 13 14 15 16 17 18 19 20

11 12 13 14 15 16 17 18 19 20

i d i b i

A B C D E F G H I

K

11 12 13 14 15 16 17 18 19 20 L M N O P Q R S T U

i d i b i

5 mm

5 mm

4 x 10 x 10 squares

Specifications :

Ordering Information:

Grid repeat distance

500 µm

Cat. No.

Description

Ø µ-Dish

35 mm

80156

2 / 0.8 ml

µ-Dish 35 mm, low, Grid-500, ibiTreat, tissue culture treated, sterile, low wall, grid repeat distance of 500 µm

60

Volume high / low Growth area

3.5 cm2

80151

µ-Dish 35 mm, low, Grid-500, hydrophobic, uncoated, sterile, low wall, grid repeat distance of 500 µm

60

81166

µ-Dish 35 mm, high, Grid-500, ibiTreat, tissue culture treated, sterile, high wall, grid repeat distance of 500 µm

60

81161

µ-Dish 35 mm, high, Grid-500, hydrophobic, uncoated, sterile, high wall, volume 2 ml, grid repeat distance of 500 µm

60

80050

DIC lid, with a glass insert, suitable for use with all ibidi 35 mm dishes, sterile

5

Coating area using 400 µl 4.1 cm

64

2

Ø observation area

21 mm

Height with lid high / low

14 / 9 mm

Bottom: ibidi Standard Bottom

Pcs. / Box


µ-Dish 35mm, high glass bottom Grid-50 µ-Dish 35mm, high glass bottom Grid-500 A 35 mm imaging dish with a glass bottom and an imprinted 50 µm / 500 µm relocation grid

• Easy cell counting per defined area • Glass coverslip for TIRF and single molecule applications

Applications • Relocating cells or cell clusters • Counting events per defined area (e.g., when calculating transfection efficiency) • Providing a reference structure for cell movements

Grid-50

• Treatment of distinct single cells, such as in microinjection • Following axon growth on a defined scale bar

45 µm

Technical Features

50 µm

• Grid with 50 µm / 500 µm repeat distance

< 5 µm

5 µm

• Lettered and numbered 1 x 400 / 4 x 400 squares (A-U; 1-20) • Grid and cells in one focal plane

Cell Counting & Relocation

• Ideal for relocating cells or cell clusters

• Bottom made from D 263 M Schott glass with a thickness of 170 µm +/- 5 µm • May require coating to promote cell attachment Layout Grid-50 Layout Grid-500 see opposite page

A i d i b i A B C D E F G H I K L M N O P Q R S T U 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 K L M N O P Q R S T U 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 K L M N O P Q R S T U

1 2 3 4 5 6 7 8 9 10

A B C D E F G H I

11 12 13 14 15 16 17 18 19 20

A B C D E F G H I

i d i b i

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

A B C D E F G H I

i d i b i

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

K

i d i b i A B C D E F G H I K L M N O P Q R S T U 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 K L M N O P Q R S T U 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 A B C D E F G H I K 20 L M N O P Q R S T U A B C D E F G H I

i d i b i

A B C D E F G H I

K

A B C D E F G H I

1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 20

11 12 13 14 15 16 17 18 19 20

K

"5 mm"

K

1 2 3 4 5 6 7 8 9 10 L M N O P Q R S T U

K

11 12 13 14 15 16 17 18 19 20 L M N O P Q R S T U

i d i b i

A B C D E F G H I

D

i d i b i A B C D E F G H I

K

A B C D E F G H I

K

A B C D E F G H I

K

1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 20

11 12 13 14 15 16 17 18 19 20

L M N O P Q R S T U

i d i b i

K

L M N O P Q R S T U 1 2 3 4 5 6 7 8 9 10

A B C D E F G H I

L M N O P Q R S T U 11 12 13 14 15 16 17 18 19 20

11 12 13 14 15 16 17 18 19 20

i d i b i A B C D E F G H I

L M N O P Q R S T U 1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

K

1 2 3 4 5 6 7 8 9 10 L M N O P Q R S T U

11 12 13 14 15 16 17 18 19 20 A B C D E F G H I

K

11 12 13 14 15 16 17 18 19 20 L M N O P Q R S T U

i d i b i

K

L M N O P Q R S T U 1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10 A B C D E F G H I

C

50 µm

i d i b i

L M N O P Q R S T U

11 12 13 14 15 16 17 18 19 20 A B C D E F G H I

L M N O P Q R S T U

i d i b i

K 1 2 3 4 5 6 7 8 9 10

A B C D E F G H I

L M N O P Q R S T U 11 12 13 14 15 16 17 18 19 20

11 12 13 14 15 16 17 18 19 20

A

i d i b i A B C D E F G H I

L M N O P Q R S T U 1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

i d i b i

A B C D E F G H I

B

i d i b i

i d i b i A B C D E F G H I K L M N O P Q R S T U 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 K L M N O P Q R S T U 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 K L M N O P Q R S T U

A B C D E F G H I

A B C D E F G H I

i d i b i A B C D E F G H I K L M N O P Q R S T U 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 K L M N O P Q R S T U 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 A B C D E F G H I K 20 L M N O P Q R S T U

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

K

1 2 3 4 5 6 7 8 9 10 L M N O P Q R S T U

11 12 13 14 15 16 17 18 19 20

11 12 13 14 15 16 17 18 19 20 A B C D E F G H I

K

11 12 13 14 15 16 17 18 19 20 L M N O P Q R S T U

i d i b i

4 x 10 x 10 squares

Ordering Information: Cat. No. 81148

Description

Specifications : Pcs. / Box

µ-Dish 35 mm, high, glass bottom Grid-50, high wall, D 263 M Schott glass, No. 1.5H (170 +/- 5 µm), sterile, grid repeat distance 50 µm

30

81168

µ-Dish 35 mm, high, glass bottom Grid-500, high wall, D 263 M Schott glass, No. 1.5H (170 +/- 5 µm), sterile, grid repeat distance 500 µm

30

80050

DIC lid, with a glass insert, suitable for use with all ibidi 35 mm dishes, sterile

5

Grid repeat distance

50 / 500 µm

Ø µ-Dish

35 mm

Volume

2 ml

Growth area

3.5 cm2

Coating area using 400 µl 4.1 cm2 Ø observation area

21 mm

Height with / without lid

14/12 mm

Bottom: Glass coverslip No. 1.5H selected quality, 170 µm +/- 5 µm

65


µ-Slide 2 well | 4 well | 8 well EW

Cell-Based Microscopy Assays

N

Open µ-Slides (chambered coverslips) with 2, 4, or 8 wells for immunofluorescence and high end microscopy

• All-in-One chamber reduces number of experimental steps in immunofluorescence protocol • Observation of the sample through the coverslip-like bottom in high resolution microscopy • Fast and easy sample preparation: No coverslips, no leakage • Cost-effective experiments using small numbers of cells and low volumes of reagents

Applications • Cultivation and microscopy of cell cultures • Fluorescence microscopy of living and fixed cells • Transfection Assays • Live cell imaging over extended time periods • Suitable for DIC, when used with a DIC lid (see page 68) Immunofluorescence of Human Umbilical Vein Endothelial Cells (HUVEC) in a µ-Slide 4 well Blue: Nucleus (DAPI) Green: VE-Cadherin (Alexa555) Red: F-Actin (Alexa488)

Technical Features • Open µ-Slide with 2, 4, or 8 independent wells • Excellent optical quality for high end microscopy • Compatible with staining and fixation • ibiTreat surface for optimal cell adhesion • Biocompatible plastic material – no glue, no leaking • Also available with a glass bottom (see page 68)

Closely fitting lid

ibidi TIP

Suitable for high resolution microscopy

Use the µ-Slide 2 well | 4 well | 8 well as the universal microscopy chamber for all applications.

100 x

Specifications : µ-Slide Number of wells

Ordering Information: 2 well

4 well

8 well

2

4

8

Dimensions of wells 21.2 x 23.3 x 9.3 21.2.x 11.0 x 9.3 9.4 x 10.7 x 6.8 (w x l x h) in mm

2 well

Coating

Cat. No.

ibiTreat, tissue culture treated, sterile

80286

4 well

8 well Pcs. / Box

80426

80826

15

Volume per well

1500 µl

700 µl

300 µl

Collagen IV, sterile

80282

80422

80822

15

Total height with lid

10.8 mm

10.8 mm

8 mm

Fibronectin, sterile*

80283

80423

80823

15

Growth area per well 4.8 cm2

2.2 cm²

1.0 cm²

Poly-L-Lysine, sterile

80284

80424

80824

15

Coating area per well 7.5 cm²

4.1 cm²

2.20 cm²

Poly-D-Lysine, sterile*

80285

80425

80825

15

hydrophobic, uncoated, sterile

80281

80421

80821

15

Bottom:

66

µ-Slide

ibidi Standard Bottom

DIC Lid for µ-Slides, sterile * available only on request

80055

5


µ-Slide 2 well Ph+ | 4 well Ph+

N

EW

Open µ-Slides (chambered coverslips) with 2 or 4 wells and a special intermediate plate for excellent phase contrast and high end fluorescence microscopy

• Excellent phase contrast over the entire well – no meniscus • All-in-one chamber slide used for cell culture and microscopy • Observation of the sample through the coverslip-like bottom in high resolution microscopy

Applications

Technical Features

• Cultivation and microscopy of cell cultures

• Open µ-Slide with 2 or 4 independent wells

• Transfection assays • Fluorescence microscopy of living and fixed cells

• Intermediate plate in each well to avoid meniscus formation

• Live cell imaging over extended time periods

Microscopy

• Fast and easy immunofluorescence, with no coverslips and no leakage

• Easy filling, without air bubbles, through the lateral slot Working with the µ-Slide 2 well Ph+ | 4 well Ph+ diminishes the meniscus and increases the area of well contrasted cells

• Excellent optical quality for high-end microscopy • Compatible with staining and fixation • Also available with a glass bottom (see page 68)

The good contrast is due to the parallel beam path that is created by the intermediate plate. Without Intermediate Plate

Ph+: With Intermediate Plate

Specifications : µ-Slide

Ordering Information: 2 well Ph+

4 well Ph+

Number of wells

2

4

Coating

Cat. No.

Dimensions of wells (w x l x h) in mm

21.2 x 23.3 x 3.0

21.2 x 11.0 x 3.0

ibiTreat, tissue culture treated, sterile

80296

80446

15

Volume per well

1500 µl

700 µl

Collagen IV, sterile

80292

80442

15

Liquid height

3.0 mm

3.0 mm

Fibronectin, sterile*

80293

80443

15

Total height with lid

10.8 mm

10.8 mm

Poly-L-Lysine, sterile

80294

80444

15

Growth area per well

4.8 cm²

2.2 cm²

Poly-D-Lysine, sterile*

80295

80445

15

Coating area per well

11.4 cm²

5.9 cm²

hydrophobic, uncoated, sterile

80291

80441

15

Bottom:

ibidi Standard Bottom

µ-Slide

DIC Lid for µ-Slides, sterile * available only on request

2 well Ph+

80055

4 well Ph+ Pcs. / Box

5

67


µ-Slide 2 well | 4 well | 8 well glass bottom µ-Slide 2 well Ph+ | 4 well Ph+ glass bottom EW

Cell-Based Microscopy Assays

N

Open µ-Slides with 2, 4, or 8 wells with a glass bottom, suitable for use in TIRF and single molecule applications

• Perfect cell imaging thanks to the low thickness variability of the coverslips’ glass • Cost-effective experiments using small numbers of cells and low volumes of reagents 100 x

Glass Bottom

• Excellent phase contrast over the entire well in Ph+ versions – no meniscus

Applications • Cultivation and microscopy of cell cultures • TIRF and single molecule applications

Technical Features ibidi Standard Bottom versus ibidi Glass Bottom: For a comparison of optical properties and other aspects please refer to page 173.

• Open µ-Slide with 2, 4, or 8 independent wells • Ph+ versions: Intermediate plate in each well to avoid meniscus formation • Bottom made from D 263 M Schott glass, No. 1.5H (170 +/- 5 µm) • May require coating to promote cell attachment

Specifications : µ-Slide Number of wells

Ordering Information: 2 well

4 well

8 well

2

4

8

Dimensions of wells 21.2 x 23.3 x 9.3 21.2 x 11.0 x 9.3 (w x l x h) in mm

9.4 x 10.7 x 6.8

Volume per well

1500 µl

700 µl

300 µl

Total height with lid

10.8 mm

10.8 mm

8 mm

Growth area per well 4.8 cm2

2.2 cm²

1.0 cm²

Coating area per well 7.5 cm²

4.1 cm²

2.20 cm²

Bottom:

Glass coverslip No. 1.5H, selected quality, 170 µm +/- 5 µm

Cat. No.

Description

80287

µ-Slide 2 well glass bottom, D 263 M Schott glass, No. 1.5H (170 +/- 5 µm), sterile

Pcs. / Box 15

80297

µ-Slide 2 well Ph+ glass bottom, D 263 M Schott glass, No. 1.5H (170 +/- 5 µm), sterile

15

80427

µ-Slide 4 well glass bottom, D 263 M Schott glass, No. 1.5H (170 +/- 5 µm), sterile

15

80447

µ-Slide 4 well Ph+ glass bottom, D 263 M Schott glass, No. 1.5H (170 +/- 5 µm), sterile

15

80827

µ-Slide 8 well glass bottom, D 263 M Schott glass, No. 1.5H (170 +/- 5 µm), sterile

15

DIC Lid for µ-Slides • For use in Differential Interference Contrast (DIC)

N EW

• Suitable for use with all ibidi µ-Slides (except Channel and Ph+ versions) Ordering Information:

68

Cat. No.

Description

80055

DIC Lid for µ-Slides, sterile

Pcs. / Box 5


sticky-Slide 8 well An 8 well bottomless µ-Slide with a self-adhesive underside to which own substrates can be mounted

• Versatile – i.e., permits the mounting of a variety of bottom materials • Has an open well format used for classical cell culture applications, which can be run on any custom surface

Microscopy

Applications • Housing for various cell culture applications • Usable with a range of bottom materials, such as plastic sheets, silicon chips, circuit boards, glass coverslips, and glass slides

Technical Features • Bottomless slide • Self-adhesive underside • Biocompatible adhesive that has been cell culture tested • Adheres to all flat surfaces, even wet surfaces • Sterile packaging • Suitable coverslips available • Removable with acetone • Specifications identical to those of the µ-Slide 8 well, except that it has no bottom Example 1: Spotted Coverslip Housing (i.e., Protein or DNA Spots)

Example 2: Circuit Board Housing

Ordering Information:

Specifications :

Cat. No.

Description

80828

sticky-Slide 8 well, sterile

10812

Coverslips for sticky-Slides, D 263 M Schott glass, No. 1.5H (170 +/- 5 µm), unsterile, 25 mm x 75 mm

Pcs. / Box 15 100

Number of wells

8

Dimensions of wells (w x l x h) in mm

9.4 x 10.7 x 6.8

Volume per well

300 µl

Total height with lid

8 mm

Growth area per well

1.0 cm2

Bottom

none

69


µ-Slide 2 x 9 well

Cell-Based Microscopy Assays

A µ-Slide suitable for co-cultivation assays in combination with microscopy

• Cells share soluble factors but grow separately • Possesses low evaporation properties and is thus suitable for long-term assays • Excellent optical quality and is therefore suitable for fluorescence microscopy

Applications ibidi APPLICATION NOTE Read our Application Note

• Co-cultivation of different cell lines or primary cells • Mesenchymal-epithelial interactions • Paracrine interaction of different cell populations in vitro • Cell or spheroid culture in gel matrices

Co-Cultivation (AN 10) for more information on how to co-cultivate two different cell types in a µ-Slide 2x9 well.

Technical Features • Two major wells each with 9 minor wells • Cells share soluble factors but grow separately

Seeding Cells Detailed Cross Section with Indicated Conditioned Medium

Step 1: Seed 60 µl of recipient cells in the inner reservoir.

Cross section Step 2: Seed 60 µl of the feeder cells in the outer reservoirs. Await cell attachment; aspirate all unattached cells and medium. Feeder cells

Step 3:

Recipient cells

Feeder cells

Overlay each major well with 600 µl cell free medium.

Specifications: Number of major wells

2

Volume per major well

600 µl

Ordering Information:

Dimensions of major wells 21.5 x (w x l x h) in mm 23.6 x 6.8

Cat. No.

Description

Number of minor wells

2 x 9

81806

µ-Slide 2 x 9 well, ibiTreat, tissue culture treated, sterile

15

Volume of each minor well 70 µl

81802

µ-Slide 2 x 9 well, Collagen IV, sterile

15

Dimensions of minor wells 6.1 x 6.8 (w x l x h) in mm x 1.3

81803

µ-Slide 2 x 9 well, Fibronectin, sterile*

15

81804

µ-Slide 2 x 9 well, Poly-L-Lysine, sterile

15

81805

µ-Slide 2 x 9 well, Poly-D-Lysine, sterile*

15

81801

µ-Slide 2 x 9 well, hydrophobic, uncoated, sterile

15

Growth area per minor well 0.4 cm2 Coating area per minor well 0.55 cm

70

Bottom: ibidi Standard Bottom

2

* available only on request

Pcs. / Box


µ-Slide I A combination of a cell culture chamber and a coverslip for imaging inside a channel

• Large observation area for microscopy • Small volume in channel • Long-term cultivation possible

• Immunofluorescence stainings with easy liquid exchange • Real-time imaging during staining process • High-resolution microscopy of living and fixed cells

Technical Features

1 mm

Microscopy

Applications

• Lids for long-term cultivation Nutrients

• See also µ-Slide I Luer (see page 128)

0.4 mm 0.18 mm

Example: Seeding Cells into a µ-Slide I O2, CO2

Step 1:

Step 2:

Step 3:

Fill the channel with 100 µl cell suspension

Fill one reservoir with cell-free medium

Fill second reservoir with the same amount of cell-free medium to equilibrate the system

Ordering Information: Pcs. / Box

Specifications: Volume per reservoir

600 µl

Number of channels

1

Volume of the channel

100 µl

Height of the channel

0.4 mm

Length of the channel

50 mm

Width of the channel

5 mm

Cat. No.

Description

80106

µ-Slide I, ibiTreat, tissue culture treated, sterile

15

80101

µ-Slide I, Collagen IV, sterile

15

80102

µ-Slide I, Fibronectin, sterile*

15

80110

µ-Slide I, Poly-L-Lysine, sterile

15

Growth area

2.5 cm2

80115

µ-Slide I, Poly-D-Lysine, sterile*

15

Coating area

5.4 cm2

80111

µ-Slide I, hydrophobic, uncoated, sterile

15

Bottom: ibidi Standard Bottom

* available only on request

µ-Slide I Luer Family Channel slides with different heights, volumes, and coatings for flow assays and high resolution microscopy

For a detailed product description and ordering information please refer to page 128.

71


micro-Insert 4 well Family

Cell-Based Microscopy Assays

A 4-well silicone insert for live cell imaging of both adherent and suspension cells

• Allows long-term microscopy assays of single cells • Cultivation of small numbers of cells without evaporation risks • Conical wells for superb optical quality Possible Filling Procedures: 10 µl

Applications • Live cell imaging of adherent and suspension cells

Minor Well Filling • Minimal volume • Individual wells

• Immobilization of cells in small wells, e.g., for 3D assays • Performing long-term microscopy assays lasting up to several weeks • Co-cultivation of cells

150 µl

• Cell tracking of very small cell numbers over time Whole Well Filling

• Cell differentiation, e.g., single stem cells

• Moderate volume • Wells connected by medium

Technical Features • Four small 2.0 mm x 1.5 mm microscopy wells with a 4:3 digital format

2 ml

• Suspension cells cannot escape the observation field • Adhesive underside

Whole µ-Dish Filling

• Transferable to any flat clean surface

• Maximum volume

• No material is left behind upon the insert’s removal

• Maximum experimental duration

• Includes a rim for easy handling of the insert with tweezers Ideal Coverage Using Digital Imaging with CCD Cameras

3D Application

micro-Insert 4 well: Rectangle well Gel filling

Whole well with cells

Digital image (tile image with overlap)

µ-Dish filling

Good coverage Standard plate: Round well

72

Bad coverage


Single micro-Insert 4 well in a 35 mm µ-Dish Ready-to-use – i.e., transfer of inserts not required

• Individual inserts for single experiments • Ideal cell growth on ibiTreat surface • An uncoated version of the µ-Dish is available, allowing for the application of individual coatings and subsequent re-insertion of the micro-Insert 4 well

Microscopy

micro-Insert 4 well 24 Ready-to-use – i.e., transfer of inserts not required

• For screening applications • High parallelization • Plate made of polystyrene – i.e., it is not suitable for either fluorescence or high resolution microscopy • An uncoated version of the 24 well plate is available for use with suspension cells

25 micro-Inserts 4 well for self-insertion For specialized use – transfer of inserts required

• 25 pieces in a transport dish • Used for self-insertion into 6 or 12 well plates, or other formats • Not to be used without transferring the micro-Inserts

Ordering Information:

Specifications:

Cat. No.

Description

80406

micro-Insert 4 well in µ-Dish 35 mm, high, ready to use, ibiTreat, tissue culture treated, sterile

80246

micro-Insert 4 well 24, a 24 well plate with 24 ready to use micro-Inserts 4 well, tissue culture treated, polystyrene, sterile

3

80245

micro-Insert 4 well 24, a 24 well plate with 24 ready to use micro-Inserts 4 well, hydrophobic, uncoated, polystyrene, sterile

3

25 micro-Inserts 4 well for self-insertion, sterile, in a 10 cm transport dish

25

80409

Pcs. / Box 30

Number of wells

4

Outer dimensions (w x l) in mm

2.0 x 1.5 (digital format 4:3)

Diameter of the complete insert

12 mm

Volume per well

10 µl

Growth area per well 0.03 cm2 Coating area per well 0.23 cm2 Material

Biocompatible silicone

Bottom

No bottom – sticky underside

73


Cell-Based Microscopy Assays

Immunofluorescence (IF) Every fluorescence protocol consists of four major steps. All these steps can be performed directly in ibidi’s µ-Slides and µ-Dishes. Main advantages are:

• Fast and simple preparation All-in-one chambers simplify your immunofluorescence protocol • Homogeneous cell distribution Cell densities are independent of handling during cell seeding • Cost-effective experiments Only a small number of cells and low volumes of reagents are needed • High resolution imaging Can be used for wide-field fluorescence, confocal imaging, FRAP – FRET – FLIM, and undisturbed phase contrast imaging

Eliminate the Need for Tweezers in IF Protocols The protocols below show that the ibidi method for performing immunofluorescence assays is superior to the traditional coverslip method. Protocol with ibidi μ-Slides (New method using all-in-one chambers)

74

Cultivation

Fixation

Staining

Imaging

Protocol with Cells on Coverslips (Traditional method with nail polish mounting)

100 x

• Sterilize coverslips and slides • Coat coverslips • Place sterile coverslips into 6-well plate • Seed cells in large volume • Peel off the coverslip • Wash • Fix – wash – permeabilize – wash - block • Wash • Stain cells FAST • Wash AND • Mount cells with mounting medium EASY • Mount coverslip with nail polish

• Sterilize coverslips and slides • Coat coverslips • Place sterile coverslips into 6-well plate • Seed cells in large volume • Peel off the coverslip • Wash • Fix – wash – permeabilize – wash – block • Wash • Stain cells TIME ING • Wash UM ONS D • Mount cells with mounting medium C AN S OU • Mount coverslip with nail polish TEDI


Technical Details Comparison of Various Immunofluorescence Applications Channel Formats – All-inOne

µ-Slide Angiogenesis

Removable Chambers

µ-Slide 8 well µ-Dish 35 mm, high

micro-Insert 4 well

12 well Chamber, removable

Volume staining reagent per well or channel

25 µl

100 µl

60 µl

100 µl

400 µl

5 µl

100 µl

Growth area per well or channel

0.6 cm²

2.5 cm²

0.125 cm2

1.1 cm²

3.5 cm²

0.03 cm²

0.56 cm²

Culture volume

150 µl

220 µl

60 µl

300 µl

2 ml

10 µl

250 µl

6

1

15

8

1

4

12

80606 (ibiTreat)

80176 (ibiTreat)

81506 (ibiTreat)

80826 (ibiTreat)

81156 (ibiTreat)

80406 (ibiTreat)

81201 (glass slide)

78

128

100

66

61

72

85

Number of independent tests Catalog number See page

Human umbilical vein endothelial cells (HUVEC) cultured under flow conditions in µ-Slide I 0.4 Luer

Cell line Madin-Darby canine kidney (MDCK) cultured in µ-Slide VI 0.4 Blue: nucleus (DAPI)

Differentiated mouse fibroblasts cultured on elastic surface (ESS 28 kPa)

Blue: nucleus (DAPI)

Green: actin cytoskeleton (Alexa 488 conjugated antibody)

Green: zyxin (Alexa 488 conjugated antibody)

Red: mitochondria (MitoTracker)

Red: alpha-smooth muscle actin (Cy5 conjugated antibody)

Green: VE-cadherins (Alexa 488 conjugated antibody) Red: actin cytoskeleton (Cy5 conjugated antibody)

(courtesy ibidi research group)

Live Immunofluorescence Cell Imaging

µ-Slide VI 0.4 µ-Slide I 0.4 Luer

Open Well Formats – All-in-One

(courtesy R. Merkel, Jülich, Germany)

(courtesy S. Zahler, Munich, Germany)

ibidi MOVIE Be part of the ibidi calendar!

View detailed handling steps in the movie:

Submit your immunofluorescence images to info@ibidi.de and place your most successful experiments on your lab’s wall.

Immunofluorescence Using the µ-Slide VI (MV 18) on www.ibidi.com

75


Cultivation

Fixation

Cell-Based Microscopy Assays

Mounting

Staining

Imaging

100 x

The ibidi channel μ-Slides are ideal for exact exchanges of small amounts of reagents, like media, buffer, washing, or staining solutions. As the channel geometries are small, the amount of staining solution required is reduced to as little as 25 μl (such as with the μ-Slide VI 0.4). In addition, the slides’ built-in channel structure dispenses with the need for additional coverslips. ibidi’s µ-Slides VI 0.4 (see page 78) are perfectly suited for general immunofluorescence assays, whereas the µ-Slide I 0.4 Luer (see page 128) is best suited for immunofluorescence assays in low density cultures and for flow experiments.

ST ED MO END M M CO RE

In addition, ibidi µ-Slides are compatible with all common staining and fixation techniques. Finally, the ibidi Mounting Medium (see page 55) prevents photobleaching and can be used to store samples for up to several weeks.

µ-Slide VI 0.4 (see page 78)

Cultivation

All-in-One Channel Formats for IF

Fixation, permeabilization, Staining blocking

Mounting

Imaging

All-in-One Open Well Formats for IF Cells are cultured in the classic open well geometry. Fixation, permeabilization, and staining steps are also done in these wells. After staining and mounting, the sample can be observed through the coverslip-like bottom using high resolution microscopy.

100 x

The ibidi μ-Slide 2 well | 4 well | 8 well (see page 66), the µ-Slide 2 well Ph+ | 4 well Ph+ (see page 67), and the μ-Dish 35 mm, high (see page 61) allow the use of standard immunofluorescence protocols, without using coverslips, in an all-in-one open well chamber.

ST ED MO END M M CO RE

Advantages of All-in-One Chambers • Fast protocol using an all-in-one chamber

µ-Slide 8 well (see page 66)

• Easy and rapid handling • No cell transfer Meniscus Formation Meniscus Formation Open OpenWell Well

Channel Channel

• No coverslip handling Ph+ Ph+

• Sterile and ready to use Limitations of All-in-One Chambers

76

In open formats, meniscus formation can be prevented by using small coverslips. In channel structures and ibidi’s Ph+ µ-Slides, this type of “lid” is integrated into the slide leading to perfect imaging quality without meniscus formation.

• Storage time is restricted to several weeks due to gas exchange through the plastic material from which the slides are made


Removable Chambers for IF

micro-Insert 4 well (see page 72)

The technique employed to use these slides is similar to the standard method. The cells are cultured in the wells of the 12 well Chamber’s silicone gasket using a culture volume of 250 µl. For immunofluorescence staining, the user can then employ any one of the protocols shown in the diagram below. 12 well Chamber, removable (see page 85)

Customers receive the product in ready-to-use form in a sterile package.

Immunofluorescence

ibidi also provides the easiest and most convenient solution for removable culture wells. These products contain a silicon gasket with chambers for individual cell culture and incubation conditions. The ibidi microInsert 4 well (see page 72) allows the user to employ extremely small volumes of medium and cell numbers for cell cultivation. The removable 12 well Chamber (see page 85) is perfect for long-term storage of samples prepared using the classical “glass slide with mounted coverslip” technique.

Different protocols possible Culture cells

Advantages of Removable Chambers • The micro-Insert 4 well requires extremely small volumes of reagents and small numbers of cells for cultivation and staining • The 12 well Chamber, removable, is perfect for long-term storage of samples • The 12 well Chamber, removable, allows the user to perfom up to 12 parallel tests on one slide

in single wells

Liquid changes for fixation permeabilization blocking

• Sterile and ready to use Limitations of Removable Chambers • The 12 well Chamber, removable, requires large volumes of reagents and high numbers of cells for cultivation and staining

ibidi APPLICATION NOTE

in single wells

Staining

in bulk ca. 20 µl

with small cover slip ca. 5 µl

Mounting for microscopy

under cover slip

Imaging

Find detailed immunofluorescence staining protocols using the µ-Slide VI 0.4 (AN 09) and the µ-Slide 8 well (AN 16) on www.ibidi.com.

ibidi FREE SAMPLES Order a free sample at www.ibidi.com to test ibidi’s µ-Slides, µ-Dishes and µ-Plates with your experiments.

77


µ-Slide VI 0.4

Cell-Based Microscopy Assays

A 6 channel μ-Slide suitable for multiple immunofluorescence stainings under both static and flow conditions

• All-in-one chamber that simplifies immunofluorescence protocols • Homogeneous cell distribution over the channel surface, regardless of handling practices • Cost-effective experiments with small numbers of cells and low volumes of reagents

Applications Nutrients Cells

• Immunofluorescence assays and live cell imaging 1 mm

• Real-time imaging under either static or flow conditions

0.4 mm

• Parallel screenings using multichannel pipettes

0.18 mm

O2, CO2 100 x

Technical Features • 30 µl channel volume, which saves on reagent consumption • Easy connection to existing tubes and pumps via female Luer adapter • Fully compatible with high resolution fluorescence microscopy • Defined shear stress and shear rate levels

All-in-One-Chamber for Fast Immunofluorescence Protocols The entire observation field is in excellent phase contrast

1) Cultivation

2) Fixation

3) Staining

4) Imaging

ibidi MOVIE View detailed handling steps in the movie: Immunofluorescence Using the µ-Slide VI (MV 18) on www.ibidi.com

100 x

Specifications: Adapters

78

Ordering Information: Female Luer

Volume per reservoir

60 µl

Number of channels

6

Volume of each channel

30 µl

Height of channels

0.4 mm

Cat. No.

Description

Pcs. / Box

80606

µ-Slide VI 0.4, ibiTreat, tissue culture treated, sterile

15

80602

µ-Slide VI 0.4, Collagen IV, sterile

15

80603

µ-Slide VI 0.4, Fibronectin, sterile*

15

µ-Slide VI 0.4, Poly-L-Lysine, sterile

15

Length of channels

17 mm

80604

Width of channels

3.8 mm

80605

µ-Slide VI 0.4, Poly-D-Lysine, sterile*

15

Growth area per channel

0.6 cm2

80601

µ-Slide VI 0.4, hydrophobic, uncoated, sterile

15

Coating area per channel

1.20 cm2

Bottom: ibidi Standard Bottom

* available only on request


sticky-Slide VI 0.4 A bottomless 6 channel slide used for cell culture applications with a self-adhesive underside to which own substrates can be mounted

Immunofluorescence - Channels

• Versatile – i.e., permits the mounting of a variety of bottom materials • Cost-effective experiments with small numbers of cells and low volumes of reagents

Applications • Running multiple immunofluorescence assays and live cell imaging • Real-time imaging under either static or flow conditions • Inserting materials or tissue into perfusion channels • Running of shear stress experiments on any substrate • Usable with a range of bottom materials, such as plastic sheets, silicon chips, and glass slides

Technical Features • Bottomless slide • Self-adhesive underside • Biocompatible adhesive that has been cell culture tested • Adheres to all flat surfaces, even wet surfaces • Female Luer adapters for connection to tubing and pump systems • Suitable coverslips available • Specifications identical to those of the µ-Slide VI 0.4, except that it has no bottom Example: Setup of a Sample Squeezed into a Channel sticky-Slide sticky-Slide with channel

Coverslip Sample

Sample preparation

Pressing and sample squeezing

Ordering Information: Cat. No. 80608 10812

Description sticky-Slide VI 0.4, sterile Coverslips for sticky-Slides, D 263 M Schott glass, No. 1.5H (170 µm +/- 5 µm), unsterile, 25 mm x 75 mm

Assembled channel

Specifications: Pcs. / Box 15 100

Adapters

Female Luer

Volume per reservoir

60 µl

Number of channels

6

Volume of each channel

30 µl

Height of channels

0.4 mm

Length of channels

17 mm

Width of channels

3.8 mm

Growth area per channel

0.6 cm2

Bottom

none

79


µ-Slide VI – flat

Cell-Based Microscopy Assays

A µ-Slide with 6 parallel channels for short term assays

• A flat all-in-one chamber for immunofluorescence assays

Applications • Immunofluorescence assays using expensive or rare reagents • Cell-based short-time assays • Parallel screening of antibodies

Technical Features • 30 µl test volume, which saves on reagent consumption • Compatible with multi-channel pipettes • No lid available; to maintain sterility for long-term assays, put the slide into a 10 cm Petri dish

100x

• Lettered and numbered channels

Condenser

• Also suitable with upright microscopes Also suitable for use with upright microscopes

Easy Immunofluorescence Protocol

1. Cultivation

2. Fixation

100x ibidi APPLICATION NOTE Find detailed protocols how to perform cell-based microscopy assays on our website:

3. Staining

4. Mounting and Imaging

www.ibidi.com.

Specifications:

80

Ordering Information:

Aperture Ø

4 mm

Cat. No.

Description

Total height

1.5 mm

80626

µ-Slide VI - flat, ibiTreat, tissue culture treated, sterile

15

Number of channels

6

Volume of each channel

30 µl

80622

µ-Slide VI - flat, Collagen IV, sterile

15

Height of channels

0.4 mm

80623

µ-Slide VI - flat, Fibronectin, sterile*

15

Length of channels

17 mm

80624

µ-Slide VI - flat, Poly-L-Lysine, sterile

15

Width of channels

3.8 mm

80625

µ-Slide VI - flat, Poly-D-Lysine, sterile*

15

Growth area per channel

0.6 cm

80621

µ-Slide VI - flat, hydrophobic, uncoated, sterile

15

Coating area per channel

1.20 cm

Bottom: ibidi Standard Bottom

2 2

* available only on request

Pcs. / Box


µ-Slide I 0.4 Luer A channel µ-Slide suitable for immunofluorescence assays in low density cultures and for flow experiments

Ordering Information: Cat. No.

Description

80176

µ-Slide I 0.4 Luer, ibiTreat, tissue culture treated, sterile

Pcs. / Box 15

80172

µ-Slide I 0.4 Luer, Collagen IV, sterile

15

80173

µ-Slide I 0.4 Luer, Fibronectin, sterile*

15

80174

µ-Slide I 0.4 Luer, Poly-L-Lysine, sterile

15

80175

µ-Slide I 0.4 Luer, Poly-D-Lysine, sterile*

15

80171

µ-Slide I 0.4 Luer, hydrophobic, uncoated, sterile

15

* available only on request

µ-Slide I A channel µ-Slide for immunofluorescence stainings with large observation area and easy liquid exchange

For a detailed product description please refer to page 71.

Immunofluorescence - Channels

For a detailed product description please refer to page 128.

Ordering Information: Cat. No.

Description

80106

µ-Slide I, ibiTreat, tissue culture treated, sterile

Pcs. / Box 15

80101

µ-Slide I, Collagen IV, sterile

15

80102

µ-Slide I, Fibronectin, sterile*

15

80110

µ-Slide I, Poly-L-Lysine, sterile

15

80115

µ-Slide I, Poly-D-Lysine, sterile*

15

80111

µ-Slide I, hydrophobic, uncoated, sterile

15

* available only on request

ibidi Mounting Medium A mounting medium that is optimized for fluorescence microscopy and ibidi’s µ-Slides and µ-Dishes

• Ready-to-use in widefield fluorescence, confocal microscopy, and immunofluorescence assays • No autofluorescence – bright fluorescence signals For a detailed product description please refer to page 55. Ordering Information: Cat. No.

Description

50001

ibidi Mounting Medium, for fluorescence microscopy, 15 ml

Pcs. / Box 1

81


µ-Slide 2 well | 4 well | 8 well µ-Slide 2 well Ph+ | 4 well Ph+ N

Cell-Based Microscopy Assays

EW

Open µ-Slides with 2, 4, or 8 wells for immunofluorescence and high end microscopy

• All-in-One chamber reduces number of experimental steps in immunofluorescence protocol • Observation of the sample through the coverslip-like bottom in high resolution microscopy • Fast and easy sample preparation: No coverslips, no leakage • Cost-effective experiments using low amounts of cells and reagents • Also available with a glass bottom (see page 68) For a detailed product description please refer to page 66 and 67. Ordering Information: µ-Slide

2 well

4 well

8 well

2 well Ph+ 4 well Ph+

Coating

Cat. No.

ibiTreat, tissue culture treated, sterile

80286

80486

80826

80296

80446

Collagen IV, sterile

80282

80482

80822

80292

80442

15

Fibronectin, sterile*

80283

80483

80823

80293

80443

15

Pcs. / Box 15

Poly-L-Lysine, sterile

80284

80484

80824

80294

80444

15

Poly-D-Lysine, sterile*

80285

80485

80825

80295

80445

15

hydrophobic, uncoated, sterile

80281

80481

80821

80291

80441

15

DIC Lid for µ-Slides, sterile

80055

5

* available only on request

µ-Dish 35 mm, high A 35 mm imaging dish with an ibidi Standard Bottom for use in immunofluorescence protocols and high resolution microscopy

• Fast protocol using an all-in-one chamber • Easy and rapid handling • No cell transfer For a detailed product description please refer to page 61. Ordering Information: Cat. No.

Description

81156

µ-Dish 35 mm, high, ibiTreat, tissue culture treated, sterile, high wall

Pcs. / Box 60

81151

µ-Dish 35 mm, high, hydrophobic, uncoated, sterile, high wall

60

Also available with a glass bottom (see page 62): 81158

µ-Dish 35 mm, high, glass bottom, D 263 M Schott glass, No. 1.5H (170 +/- 5 µm), sterile

60

For immunofluorescence on an imprinted relocation grid (see page 64):

82

Also available with a relocation grid

81166

µ-Dish 35 mm, high, Grid-500, ibiTreat, tissue culture treated, sterile, high wall, grid repeat distance of 500 µm

60

81161

µ-Dish 35 mm, high, Grid-500, hydrophobic, uncoated, sterile, high wall, volume 2 ml, grid repeat distance of 500 µm

60


µ-Slide Angiogenesis A µ-Slide primarily used to investigate angiogenesis. Also perfect for immunofluorescence staining and 3D cell culture.

• Can be used in a wide range of applications For a detailed product description please refer to page 100.

Normal use – for tube formation assay

Lower volume of gel – for focussing cells Collagen IV Fibronectin Laminin

Cells embedded in gel matrix – 3D cell culture

Without any gel matrix – low volume microscopy chamber

Concentration

Also applicable for IF-based coating tests

Ordering Information: Cat. No.

Description

Pcs. / Box

81506

µ-Slide Angiogenesis, ibiTreat, tissue culture treated, sterile

15

81501

µ-Slide Angiogenesis, hydrophobic, uncoated, sterile

15

81531

For Microdissection only: µ-Slide Angiogenesis, Microdissection, PEN-membrane, sterile

15

Immunofluorescence – Open Wells

• Brilliant visualization without meniscus formation, and with all cells in one focal plane

micro-Insert 4 well Family A 4 well silicone insert for cell cultivation and immunofluorescence staining

• Requires extremely small volumes of reagents and small numbers of cells • Conical wells for superb optical quality For a detailed product description please refer to page 72.

Ordering Information: Cat. No.

Description

Pcs. / Box

80406

micro-Insert 4 well in µ-Dish 35 mm, high, ready to use, ibiTreat, tissue culture treated, sterile

30

80246

micro-Insert 4 well 24, a 24 well plate with 24 ready to use micro-Inserts 4 well, tissue culture treated, polystyrene, sterile

3

80245

micro-Insert 4 well 24, a 24 well plate with 24 ready to use micro-Inserts 4 well, hydrophobic, uncoated, polystyrene, sterile

3

80409

25 micro-Inserts 4 well for self-insertion, sterile, in a 10 cm transport dish

25

ibidi TIP The micro-Insert 4 well can also be transferred to all flat and clean surfaces for special experimental setups

83


µ-Slide 18 well – flat

Cell-Based Microscopy Assays

A µ-Slide with 18 wells for use in matrix tests and quick immunofluorescence stainings

• Low volume screening slide for medium throughput • Cost-effective experiments using small numbers of cells and low volumes of reagents

Applications • Quick immunofluorescence staining of adherent cells • Optimization of both surface functionalizations and coatings • Fast toxicological screening of small microscopy samples • Spotting samples such as RNA assays • Cultivation of small organisms

Technical Features To prevent evaporation during long-term assays place the slides in an Olaf Humidifying Chamber.

• Lettered and numbered wells (A-C; 1-6) • Suitable for fluorescence scanners • Excellent optical quality for fluorescence microscopy

NOTE:

• Has a very small open volume and thus suffers from high evaporation rates, i.e., it is only suitable for short-term experiments*

Also available with a 1 µm foil for Laser Microdissection applications

* For longer experiments use the 15 well µ-Slide Angiogenesis

Fully compatible with multipipettes (detach the two outer pipette tips)

For multiple immunofluorescence stainings or toxicological screenings

Specifications:

Ordering Information:

To test cell attachment, coat the surfaces with different cell adhesion factors, followed by cell seeding and incubation.

Number of wells

18

Cat. No.

Description

Height with / without lid

5.0 / 1.6 mm

81826

µ-Slide 18 well - flat, ibiTreat, tissue culture treated, sterile

15

Volume per reservoir

30 µl

81822

µ-Slide 18 well - flat, Collagen IV, sterile

15

Well diameter

5 mm

81823

µ-Slide 18 well - flat, Fibronectin, sterile*

15

Growth area per well

0.2 cm2

81824

µ-Slide 18 well - flat, Poly-L-Lysine, sterile

15

Coating area per channel

0.25 cm

81825

µ-Slide 18 well - flat, Poly-D-Lysine, sterile*

15

81821

µ-Slide 18 well - flat, hydrophobic, uncoated, sterile

15

81831

µ-Slide 18 well - flat, Microdissection, PEN-membrane, sterile

15

Bottom: ibidi Standard Bottom

84

• Compatible with multichannel pipettes

2

* available only on request

Pcs. / Box


12 well Chamber, removable A removable silicone cultivation chamber for cell culture and immunofluorescence staining

• Cost effective classical immunofluorescence assays

Applications • Immunofluorescence assays using standard coverslip techniques • Long-term sample storage • 12 fold sample preparation of adherent cells and tissue samples

Technical Features • Self-adhesive 12 well silicone gasket mounted on a standard glass slide • Biocompatible silicone material • Non-fluorescent glass slide with frosted ends for easy handling and labeling Triple immunofluorescence staining of MDCK cells (blue-nucleus, green-F-actin cytoskeleton, red-mitochondria)

• Suitable for use with upright and inverted microscopes, after staining and mounting with a coverslip • Compatible with all common mounting media

Immunofluorescence – Open Wells

• Perfect for immunofluorescence staining and long-term sample storage

• Optional: The gasket may be transferred to any flat clean surface for cell cultivation

Procedure:

Ordering Information: Cat. No.

Description

Specifications: Pcs. / Box

81201

12 well Chamber, removable , microscopy glass slide, sterile

15

10811

Coverslips for 12 well Chamber, removable , D 263 M Schott glass, No. 1.5H (170 +/- 5 µm), unsterile, 24 mm x 60 mm,

100

Number of wells

12

Dimensions of wells 7.5 x 7.5 x 8 (w x l x h) in mm Volume per well

250 µl

Growth area per well

0.56 cm²

Coating area per well 1.90 cm2 Material

Biocompatible silicone

Suitable coverslip size 24 mm x 60 mm Bottom

Glass slide (26 x 76 x 1 mm)

85


Screening

Cell-Based Microscopy Assays

Tube Formation Assays in the ibidi µ-Plate Angiogenesis 96 well By utilizing ibidi’s µ-Plate Angiogenesis 96 well (see page 101) the anti-angiogenic potential of substances can be tested with functional tube formation assays. With this fully SBS (Society for Biomolecular Screening) compliant and robotics compatible screening plate, compounds can be tested for their influence on structural functionality, using an in vitro assay. The most significant example of such an assay, i.e., the functional tube formation assay, using endothelial cells on Matrigel™*, is described below.

Input:

Output:

• Endothelial cells (10,000 per well)

• Extent of tube formation per compound

• Matrigel (10 µl per well) • Medium, compounds (70 µl per well)

The µ-Plate Angiogenesis 96 well uses the proven “well-in-a-well” feature to avoid meniscus formation in individual wells.

Cell medium (70 µl)

Other advantages include: • 90 % less Matrigel is needed, compared to using standard 96-well plates • Allows for efficient imaging as all cells are in one focal plane

8000

6000

4000

2000

Compound 96

Compound 5

Compound 4

Compound 3

Compound 2

Control

Compound 95

........

0 Compound 1

Tube Length [µm*]

* Lenght in µm / Standard Area (=1 cm²)

Gel matrix (10 µl)

Furthermore, only four steps are needed to go from sample preparation to image analysis (see page 152).

Typical Screening Data Obtained with the µ-Plate Angiogenesis 96 well

Gel matrix

2. Acquisition of microscopy images

86

* Matrigel is a registered trade mark of Becton Dickinson.

4000

2000

3. Quantitative image analysis

Compound 96

Compound 5

Compound 95

........

0

Compound 4

provides practical advice on how to run tube formation assays using the µ-Plate Angiogenesis 96 well.

6000

Control

Tube Formation in µ-Plate Angiogenesis 96 well (AN05)

8000

Compound 3

The Application Note

Compound 2

Tube Length [µm*]

ibidi APPLICATION NOTE

Compound 1

1. Cell seeding and tube formation

* Lenght in µm / Standard Area (=1 cm²)

The tube length was measured after 6 hours. The graph shows that different inhibitors act on the mechanism of tube formation in varying degrees and inhibit the formation of characteristic patterns.

4. Data analysis and evaluation


Impedance-Based Wound Healing Assays using ECIS With ECIS’s impedance measurements, functional wound healing assays can be performed with unrivalled convenience. After wounding cells on an electrode with an electric pulse, ECIS’s labelfree impedance measurement technology allows the user to measure the wound healing process in real time. ECIS standard 96 well screening plates are designed for high-throughput applications, such as screening of migration inhibitors.

Input:

Output:

• Cells (50,000 per well)

• Time taken for wound healing per compound

• Medium, compounds (300 µl per well)

Wounding

10

15

20

Time [h]

14 12 10

......

Compound 96

5

Compound 95

0

16

Compound 7

1000

18

Compound 6

2000

20

Compound 5

Inhibitor

Compound 4

Normal

Compound 3

Enhancer

3000

Compound 2

4000

22

Control

5000

Compound 1

Impedance (Ohm)

Control

6000

Duration Wound Healing [h]

24

7000

0

Live Cell Screening Imaging

The general effects of enhancers and inhibitors on wound healing are provided below, along with typical data from migration inhibitors screenings.

Typical Migration Data Obtained Using ECIS Technology The emptied electrode is re-covered with cells after 14 hours. The graph shows that different inhibitors slow down the wound healing process. The ECIS principle is explained in detail starting from page 138.

Screening in ibidi’s Multi-Well Plates The ibidi technology used with µ-Plates 24 well, µ-Plates 96 well, and µ-Plates 384 well (see page 89) provides all the important features required for enhanced microscopy: a coverslip like bottom of standard No.1.5 thickness with extremely low autofluorescence, and the unique ibiTreat tissue culture treatment that makes the µ-Plates biocompatible, as proven in ibidi’s µ-Slides and µ-Dishes. Another outstanding feature of ibidi’s µ-Plates is their excellent stability with all common solvents used in cell-based assays.

ibidi FREE SAMPLES Order a free sample at www.ibidi.com to test ibidi’s Multi-well Plates with your experiments.

87


µ-Plate Family

• Ideal for cell microscopy • Excellent inner well flatness and whole plate flatness • Low well-to-well crosstalk in fluorescence microscopy

Applications • Fluorescence-based imaging of living or fixed cells 127.7

11.60

11.56 7.25 1.60

• High throughput screening (HTS) in cell culture .90

R0

• Compound screenings (toxicology) • Large-scale transfection experiments

8.40

7.25

85.5

1.60

8.40

20

. R1

Cell-Based Microscopy Assays

Black multi-well plates for high throughput applications in cell-based assays

Technical Features

ibidi TIP

• Standard format, and dimensions of 85.5 x 127.5 mm (meets ANSI / SBS Standards)

Technical drawings and details are available in the instructions on www.ibidi.com

• 24 round or 96 / 384 quadratic wells with standard numbering • Compatible with automation equipment • Excellent inner well flatness and whole plate flatness • Suitable for fluorescence scanners • High-quality plastic bottom, which is compatible with solvents for staining and fixation, and also with immersion oil • Sterile, single packaging

< 5 µm

Inner well accuracy

Whole plate accuracy A-1

D-7

H-12 < 25 µm

Excellent for Cell Microscopy Inner well flatness < 5 µm Whole plate flatness < 25 µm < 5 µm

88

Well 1

Well 12

< 25 µm


µ-Plate 24 well A black 24 well plate with flat and clear bottom for high throughput applications in cell-based assays

Ordering Information: Cat. No. Description

Specifications: Pcs. / Box

µ-Plate 24 well, ibiTreat, 15 tissue culture treated, sterile

82401

µ-Plate 24 well, hydrophobic, uncoated, sterile

15

127.7 / 85.5 mm

Coating area per well

4.3 cm2

Height with /without lid 22.5 / 20.5 mm

Volume single well

1 ml

Single well depth

18.5 mm

Inner well flatness

± 10 μm

Well-to-well distance

19.0 mm

Whole plate flatness

± 25 μm

Ø of single wells

15.5 mm

Well clearance

1.5 mm

Growth area per well

1.9 cm2

Bottom: ibidi Standard Bottom

Screening

82406

Length / Width

µ-Plate 96 well A black 96 well plate with flat and clear bottom for high throughput applications in cell-based assays

Ordering Information: Cat. No. Description

Specifications: Pcs. / Box

89626 µ-Plate 96 well, ibiTreat, 15 tissue culture treated, sterile 89621 µ-Plate 96 well, hydrophobic, 15 uncoated, sterile

Length / Width

127.7 / 85.5 mm

Coating area per well

2.35 cm2

Height with /without lid 17.2 / 15.0 mm

Volume single well

300 µl

Single well depth

13.0 mm

Inner well flatness

± 5 μm

Well-to-well distance

9.1 mm

Whole plate flatness

± 25 μm

Single well dimensions 7.3 x 7.3 mm

Well clearance

1.0 mm

Growth area per well

Bottom: ibidi Standard Bottom

0.55 cm

2

µ-Plate 384 well A black 384 well plate with a flat and clear bottom for high throughput applications in cell-based assays

Ordering Information: Cat. No. Description

Specifications: Pcs. / Box

88401 µ-Plate 384 well, hydrophobic, 15 uncoated, sterile

Length / Width

127.7 / 85.5 mm

Coating area per well

0.8 cm2

Height with / without lid 17.2 / 15.0 mm

Volume single well

50 µl

Single well depth

12.7 mm

Inner well flatness

± 5 µm

Well-to-well distance

4.55 mm

Whole plate flatness

± 25 µm

Single well dimensions 3.4 x 3.4 mm

Well clearance

1.0 mm

Growth area per well

Bottom: ibidi Standard Bottom

0.11 cm²

89


Cell-Based Microscopy Assays

Customer Feedback

“The ibidi platforms have opened new possibilities for our group to utilize small volumes of blood to characterize the cell biology of platelets and white blood cells. Moreover, we have greatly benefited from collaborating with the technical representatives at ibidi to discuss ways to multiplex the ibidi platforms to address our experimental questions.” Owen McCarty, PhD Associate Professor Department of Biomedical Engineering Oregon Health & Science University Portland, Oregon USA

“The 35 mm µ-Dish is invaluable for high resolution imaging performed in support of my work. The optical plastic is fantastic substrate for building compartmentalized neuronal cultures. Thank you for making great products.” Matthew Taylor Post-doctoral Fellow Department of Molecular Biology Princeton University Princeton, NJ USA

“I plated and transfected cells on the ibidi µ-Slide 8 well alongside the regular chambers we normally use and they were very good. In fact, I particularly liked the lids which seem more secure than our regular 8-well chambers and the fact that they are ‘slide’ sized with space for writing a label. Another bonus was the individual packaging which gives me greater confidence on sterility particularly when not all the chambers in a pack of 8 are used at once. So all in all great.” Joanne Marrison Imaging and Cytometry Laboratory (B/K051) Bioscience Technology Facility, Department of Biology University of York, York United Kingdom

References Fission yeast Ags1 confers the essential septum strength needed for safe gradual cell abscission J. C. G. Cortés, M. Sato, J. Munoz, M. B. Moreno, J. A. ClementeRamos, M. Ramos, H. Okada, M. Osumi, A. Durán and J. C. Ribas The Journal of Cell Biology, 2012, 10.1083/jcb.201202015 Activation-Induced B Cell Fates Are Selected by Intracellular Stochastic Competition K. R. Duffy, C. J. Wellard, J. F. Markham, J. H. S. Zhou, R. Holmberg, E. D. Hawkins, J. Hasbold, M. R. Dowling and P. D. Hodgkin Science, 2012, 10.1126/ science.1213230

90

Golden Goal collaborates with Flamingo in conferring synaptic-layer specificity in the visual system S. Hakeda-Suzuki, S. Berger-Müller, T. Tomasi, T. Usui, S. Horiuchi, T. Uemura and T. Suzuki Nature Neuroscience, 2011, 10.1038/nn.2756 The circadian molecular clock creates epidermal stem cell heterogeneity P. Janich, G. Pascual, A. MerlosSuarez, E. Batlle, J. Ripperger, U. Albrecht, K. Obrietan, L. Di Croce and S. A. Benitah Nature, 2011, 10.1038/ nature10649

Systematic analysis of human protein complexes identifies chromosome segregation proteins J. R. A. Hutchins, Y. Toyoda, B. Hegemann, I. Poser, J. K. Heriche, M. M. Sykora, M. Augsburg, O. Hudecz, B. A. Buschhorn and J. Bulkescher Science, 2010, 10.1126/ science.1181348


CELL CULTURE UNDER SHEAR STRESS . . 114

Culture-Insert Family . . . . . . . . . . . . . . . . . . . . 94

ibidi Pump System . . . . . . . . . . . . . . . . . . . . . 118

Single Culture-Insert in a 35 mm µ-Dish . . . . . 95

Fluidic Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Culture-Insert 24 . . . . . . . . . . . . . . . . . . . . . . . 95

Perfusion Set . . . . . . . . . . . . . . . . . . . . . . . . . . 119

25 Culture-Inserts for self-insertion . . . . . . . . 95

PumpControl Software . . . . . . . . . . . . . . . . . . 119

WimScratch – Wound Healing Image Analysis 96

Fluidic Unit Quad . . . . . . . . . . . . . . . . . . . . . . . 119

ibidi Heating & Incubation System . . . . . . . . . 96

Heating Insert Adapter for Perfusion Assay . . 119 KD Scientific Syringe Pumps . . . . . . . . . . . . . 126

ANGIOGENESIS . . . . . . . . . . . . . . . . . . . . . . 98

µ-Galaxy Incubator . . . . . . . . . . . . . . . . . . . . . 127

µ-Slide Angiogenesis . . . . . . . . . . . . . . . . . . . 100

µ-Slide I Luer Family . . . . . . . . . . . . . . . . . . . 128

µ-Plate Angiogenesis 96 well . . . . . . . . . . . . 101

sticky-Slide I Luer . . . . . . . . . . . . . . . . . . . . . 129

WimTube – Tube Formation Image Analysis . 102

µ-Slide VI 0.1 | µ-Slide III 0.1 . . . . . . . . . . . . . . . 130

WimSprout – Sprouting Image Analysis . . . . 102

µ-Slide VI 0.4 | µ-Slide III 0.4 . . . . . . . . . . . . . . . . 131

ibidi Heating & Incubation System . . . . . . . . 102

µ-Slide y-shaped . . . . . . . . . . . . . . . . . . . . . . 132 Elbow Luer Connector . . . . . . . . . . . . . . . . . . 133

CHEMOTAXIS . . . . . . . . . . . . . . . . . . . . . . . 104 µ-Slide Chemotaxis Family . . . . . . . . . . . . . . 108 µ-Slide Chemotaxis 3D . . . . . . . . . . . . . . . . . . 109 µ-Slide Chemotaxis 2D . . . . . . . . . . . . . . . . . . 109 sticky-Slide Chemotaxis 3D . . . . . . . . . . . . . . . . 110 µ-Slide III 3in1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 WimTaxis – Chemotaxis Image Analysis . . . . . 112 Chemotaxis and Migration Tool . . . . . . . . . . . . 112 Collagen Type I . . . . . . . . . . . . . . . . . . . . . . . . 112

Luer Connector Male . . . . . . . . . . . . . . . . . . . 133 Luer Lock Connector Male . . . . . . . . . . . . . . 133

Functional Cell-Based Assays

WOUND HEALING AND MIGRATION . . . . . 92

Luer Lock Connector Female . . . . . . . . . . . . 133 Female Luer Lock Coupler . . . . . . . . . . . . . . 134 Luer Plug Male . . . . . . . . . . . . . . . . . . . . . . . 134 In-line Luer Injection Port . . . . . . . . . . . . . . . 134 Hose Clip . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Tube Adapter Set . . . . . . . . . . . . . . . . . . . . . . 134 Serial Connector . . . . . . . . . . . . . . . . . . . . . . 135 Y Tube Fitting 0.8 mm / 1.6 mm . . . . . . . . . . . 135 Fitting Reducer 0.5 to 1.6 . . . . . . . . . . . . . . . 135 Silicone Tubing . . . . . . . . . . . . . . . . . . . . . . . . 135

91


0h

Experimental Workflow

Functional Cell-Based Assays

Wound Healing and Migration 6h

12 h

18 h

24 h

Sample Preparation

Microscopy

Culture-Inserts (page 94)

Microscope with Incubation (page 15)

Easy Handling

Principle

1. Prepare the Culture-Insert on a flat, clean surface

2. Seed cells and wait for cell attachment

3. Remove Culture-Insert

4. Fill with medium

When placed on a cell culture surface, the Culture-Insert provides two cell culture reservoirs which are separated by a 500 µm thick wall. The cells are cultured in both reservoirs and then the silicone insert is removed from the surface. This results in two precisely defined cell patches, which are separated by a zone that is exactly the same width as the separation wall. Due to the specially designed bottom, the CultureInsert sticks itself to the surface. This adhesive design completely prevents any cell growth under the walls. After removal from the surface, the newly created cellfree gap (wound) will be clean and unchanged, and no material will remain. With this method, you can create highly defined regions without any cells.

Easy Data Acquisition Microscopy is used to evaluate the wound healing process. Depending on your focus of interest, this can either be done by using video microscopy, or by observing images at distinct time points (e.g., after 6 and 12 hours).

5. Microscopy of cell-free gap

Cell Covered Area [µm²]

Experimental Examples 3

600x10

Enhancer Normal

500

Inhibitor

400

300

Add drug 0

5

10

15

20

25

Time [h]

92

Influence of inhibitors or enhancers on wound healing

Two different cell types (2D invasion assay) Data provided by C. Matern and K. D. Nnetu, University Leipzig, Germany

Co-cultivation assays with different cell types


35

Cell Front Velocity [µm/h]

Cell Covered Area [µm²]

600x10³

500

400

300

30 25 20 15 10 5

Image Analysis

2

1

er

er

nc

nc

ha

ha

En

r1

r2

ro

to

to

bi

bi

hi

hi

In

25

In

20

En

15

Time [h]

nt

10

Co

5

l

0 0

Quantitative and Statistical Analysis

Easy Data Analysis

Comparison to Scratch Assay

Our Wound Healing Image Analysis solution evaluates 2D cell migration. The web-based automated WimScratch software generates results from your collected data within minutes. Simply upload your data to the image analysis platform and instantly receive results via E-mail. Analysis data contain: • Cell covered area • Speed of closure (available ≥ 5 images) • Acceleration characteristics (available ≥ 5 images) • Overview chart • Center piece approximation (available ≥ 10 images)

Wound Healing Chemotaxis & Migration

Wimasis WimScratch (page 156)

Experimental Endpoints • Wound healing and migration behavior of cells and mutants (knockout / knockdown) • Wound healing potential of substances • Effect of inhibitors / enhancers to wound healing • Molecular mechanisms visualized by high resolution fluorescence microscopy

The ibidi Culture-Insert provides improved reproducibility, compared to a Scratch Assay. Data provided by M. Börries, University Freiburg, Germany.

• Investigation of signal transduction or cytoskeletal effects

ibidi MOVIE For practical details please watch our movies Performing a Wound Healing Assay (MV 16) and Wound Healing Assay Evaluated with the Wimasis Automated Image Analysis (MV 22) on www.ibidi.com.

93


Culture-Insert Family Silicone inserts with a defined cell-free gap, suitable for wound healing, migration assays, 2D invasion assays, and co-cultivation of cells

Functional Cell-Based Assays

• Complete solution for wound healing experiments, requiring only a few steps to go from sample preparation to image analysis • Reproducible experiments owing to: A defined 500 µm cell free gap, no leakage during cultivation, and no material being left behind after the insert’s removal

Applications • Wound healing assays Various Applications A ) 2D Invasion Assays Cell type I

• Migration assays • 2D invasion assays • Co-cultivation of cells

Details

Cell type II

Principle for Wound Healing and Migration Assays

B ) Co-Cultivation of Cells

Step 1: cell seeding and attachment Step 2: remove insert after cultivation Step 3: overlay cell patches with culture medium

Culture-Inserts versus Scratch Assay: Two assays that are not 100% comparable ibidi Culture-Inserts

Scratch Assay

Cell-seeding into designated areas

Scratching the cell surface with a needle or pipet tip

Defined cell-free gap

Varying cell-free gap – i.e., not reproducible

Defined non-coated surface (i.e., ibiTreat or uncoated)

Might contain extra-cellular matrix remains

No cell damage

Cell damage

Internal reference*

No internal reference* *Internal Reference The internal reference may answer the question as to whether two opposite cell fronts influence each other or not. With the defined cell seeding technique, it is possible to measure the speed of: A: a cell front that is opposite another cell front; and

94

A: Two opposite cell fronts B: Single cell front

B: a single cell front that does not have an opposite cell front.


Single Culture-Insert in a 35 mm µ-Dish Ready-to-use – i.e., transfer of inserts not required

• Individual inserts for single experiments • Ideal cell growth on ibiTreat surface • An uncoated version of the µ-Dish is available, allowing for the application of individual coatings

Wound Healing & Migration

Culture-Insert 24 Ready-to-use – i.e., transfer of inserts not required

• For screening applications • High parallelization • Plate made of polystyrene – i.e., it is not suitable for either fluorescence or high resolution microscopy

25 Culture-Inserts for self-insertion For specialized use – transfer of inserts required

• 25 pieces in a transport dish • Used for self-insertion into 6 or 12 well plates, or other formats • Not to be used without transferring the Culture-Inserts

Ordering Information: Cat. No.

Description

Specifications: Pcs. / Box

80206

Culture-Insert in µ-Dish 35 mm, low, Culture-Inserts ready-to-use in a µ-Dish 35 mm, low wall, ibiTreat, tissue culture treated, sterile

30

81176

Culture-Insert in µ-Dish 35 mm, high, Culture-Inserts ready-to-use in a µ-Dish 35 mm, high wall, ibiTreat, tissue culture treated, sterile

30

80241

80209

Culture-Insert 24, a 24 well plate with 24 ready to use CultureInserts, tissue culture treated, polystyrene, sterile 25 Culture-Inserts for self-insertion, sterile, in a 10 cm transport dish

Number of wells

2

Outer dimensions (w x l x h) in mm

8.4 x 8.4 x 5

Volume per well

70 µl

Growth area per well 0.22 cm² Coating area per well 0.82 cm2

3

25

Width of cell free gap 500 µm +/- 50 µm Material

Biocompatible silicone

Bottom

No bottom sticky underside

95


in cooperation with

WimScratch – Wound Healing Image Analysis A web-based quantitative image analysis of wound healing and cell migration assays

This Wound Healing Image Analysis solution (see page 156) evaluates 2D cell migration and generates results within minutes. No extra hardware or software is required. Simply upload your data to the image analysis platform on www.ibidi.com and instantly receive your results via E-mail.

Cell Front Velocity [µm/h]

30 25 20 15 10 5

Analysis Data Contains:

0 Co nt ro In hi l bi to In r1 hi bi En tor 2 ha n En cer 1 ha nc er 2

• Cell-covered area • Speed of closure (available ≥ 5 images)

Statistical analysis of a complete experimental series

• Acceleration characteristics (available ≥ 5 images) • Overview chart • Center piece approximation (available ≥ 10 images)

Only 4 Steps From Sample Preparation to Image Analysis 100

Cell covered area [%]

Functional Cell-Based Assays

35

95 90 85 80 75 0

20

• Cell covered area • Speed of closure • Acceleration characteristics • Overview chart 40

60

80

100

Time [h]

1. Cell seeding and gap formation

2. Acquisition of microscopy images

3. Quantitative image analysis

4. Data analysis and evaluation

ibidi Heating & Incubation System Live Cell Imaging with incubator conditions directly on the microscope

Migration and proliferation assays require full incubator conditions on the microscope for time lapse video microscopy. The ibidi Heating & Incubation System provides precise and reliable temperature control and gas incubation and a perfect illumination of the sample. It is compatible with all inverted microscope models. Humidity sensor

Heated lid

Gas inlet

For a detailed product description please refer to page 15.

Heated plate

96

Objective lens

The ibidi Gas Incubation System provides both humid and CO2rich air for stage-top incubators like the ibidi Heating System. The gas mixture is continuously flushed through the stage top incubator, ensuring a maximum humidity and an optimal pH for CO2-buffered liquids.


Customer Feedback

Judith Cathcart Advanced Optical Microscopy Facility University Health Network Toronto Canada

“The ibidi CultureInserts for wound healing assays provide a new line of testing for us. We wouldn’t do traditional scratch assays at all because of the inherent variation, but with the new inserts we’re able to provide many images to support our research. Our results are repeatable and reliable regardless of which researcher conducts the assay.” Jason W. Bjork 3M Research Materials Laboratory St. Paul USA

“The ibidi cell migration Culture-Inserts enabled me to perform highly reproducible and quantitative migration experiments. Since physical scratching isn’t required, uniformity is maintained and adverse wounding responses are not an issue. The online quantification tool WimScratch for the assay is also fantastic and it yields quantitative data from the images extremely quickly with little effort required. I’d definitely recommend it for migration studies.“ Ross Flockhart, PhD Stanford School of Medicine Department of Dermatology Stanford USA

Wound Healing & Migration

"The ibidi chambers (Culture-Inserts) we used in our wound healing assays helped to keep the cell free area clean and consistent in size without damaging the ECM or leading edge of the cell fronts, making analysis of results more manageable and comparable. They're convenient and easy to use and their small size helps to conserve precious cells and culture materials. We'd certainly use them again.“

References Leucine-rich repeat kinase 2 functionally interacts with microtubules and kinasedependently modulates cell migration M. Caesar, S. Zach, C. B. Carlson, K. Brockmann, T. Gasser and F. Gillardon Neurobiology of Disease, 2013, 10.1016/j.nbd.2012.12.019,

Regulation of ovarian cancer progression by microRNA-187 through targeting Disabled homolog-2 A. Chao, C. Y. Lin, Y. S. Lee, C. L. Tsai, P. C. Wei, S. Hsueh, T. I. Wu, C. N. Tsai, C. J. Wang and A. S. Chao Oncogene, 2011, 10.1038/ onc.2011.269

Modulation of Chemotactic and Pro-Inflammatory Activities of Endothelial Progenitor Cells by Hepatocellular Carcinoma Y.-T. Shih, M.-C. Wang, H.-H. Peng, T.-F. Chen, J.-Y. Chang and J.-J. Chiu Cellular Signalling, 2012, 10.1016/j.cellsig.2011.11.013

The carboxyl tail of Cx43 augments p38 mediated cell migration in a gap junctionindependent manner J. Behrens, P. Kameritsch, S. Wallner, U. Pohl and K. Pogoda European Journal of Cell Biology, 2010, 10.1016/j.ejcb.2010.06.003

Protease-Activated Receptor-1 Antagonist F 16618 Reduces Arterial Restenosis by Down-Regulation of Tumor Necrosis Factor ?; and Matrix Metalloproteinase 7 Expression, Migration, and Proliferation of Vascular Smooth Muscle Cells P. Chieng-Yane, A. Bocquet, R. Letienne, T. Bourbon, S. Sablayrolles, M. Perez, S. N. Hatem, A.-M. Lompre, B. Le Grand and M. David-Dufilho The Journal of Pharmacology and Experimental Therapeutics, 2011, 10.1124/jpet.110.175182

97


98

0 h

Experimental Workflow

Functional Cell-Based Assays

Angiogenesis 6 h

Sample Preparation

Microscopy

µ-Slide Angiogenesis (page 100)

Microscope with or without Incubation (page 15)

ibidi’s „Well-in-a-Well“ Compared to a Standard Well

1)

Easy Handling The ibidi tube formation assay was primarily developed to simplify the assay of endothelial cells on Matrigel™. Using a geometrical trick called “well-in-a-well“, the amount of Matrigel needed is reduced to 10 µl per well. Additionally, the gel forms a flat surface (no meniscus), which provides all the cells in one optical plane. After the gel is filled in and given time to solidify, the cells can be seeded on top and tube formation can be analyzed.

1) 2)

ibidi provides two different types of plates that contain this special geometry for angiogenesis assays (e.g., the tube formation assay). The first is the µ-Slide Angiogenesis, with 15 wells (see page 100), for low throughput applications. The second, for larger screening applications, is ibidi’s new µ-Plate Angiogenesis 96 well (see page 101).

2)

10x

2 h

10x

µ-Plate Angiogenesis 96 well

Standard well

1) Planar air-liquid interface: good phase contrast all over the observation area

1) Meniscus on airliquid interface: poor phase contrast in most of the observation area

2) Planar gel surface: all cells are in one optical plane

2) Mensicus on the gel surface: not possible to focus on all cells simultaneously

Volume of Matrigel: 10 µl

Volume of Matrigel: 100 µl

Easy Data Acquisition Microscopy is used to evaluate the tube formation process. Depending on your focus of interest, this can either be done by using video microscopy (a continuous movie), or by observing images at distinct time points (e.g., after 6 hours and 12 hours). The ibidi Heating & Incubation Systems (see page 15) enable live cell imaging with incubator conditions directly on the microscope.

One well of the µ-Slide Angiogenesis, with HUVEC cells on Matrigel after 24 hours of incubation. Stitched image, 5 x 5 mm.


Image Analysis

Tube Characteristics Total Tube Length [px] Total Tubes Mean Tube Length [px] Std. Dev. Tube Length [px]

12916 98 132 82

Loop Characteristics Total Loops Mean Loop Area [px] Std. Dev. Loop Area [px] Mean Loop Perimeter [px] Std. Dev. Loop Perimeter [px]

Tube Length [µm*]

Tube length [µm/mm²]

10x10³

8

6

4

0

24 23809 27614 614 427

2

4

6

8

Time [h]

10

* Lenght in µm / Standard Area (=1 cm²)

36 12916 58 24 1

8000

6000

4000

2000

0

Co nt ro l 1 ng /m l 10 ng /m l 10 0 ng /m l 1 µg /m l 10 µg /m l

WimTube Key Metrics Covered Area % Total Tube Length [px] Total Branching Points Total Loops Total Nets

Concentration

Quantitative and Statistical Analysis

Wimasis WimTube (page 152)

Experimental Examples

For the tube formation assay, we provide an easy-touse analysis solution. The web-based automated tool WimTube (see page 152) generates results from your data within minutes. No extra hardware or software is needed. Simply upload your data to the image analysis platform and instantly receive results via E-mail. Analysis data contains: • Total tube lengths, numbers, and statistics • Loop numbers, areas, and perimeters

Influence of inhibitors on tube formation over time. Please note the time dependence of a tube formation and take images at identical time points.

• Cell-covered area • Branching points

Angiogenesis Chemotaxis

Easy Data Analysis

Experimental Endpoints • Angiogenic potential of substances • Effect of inhibitors or enhancers on tube formation assays • Molecular mechanisms visualized by fluorescence microscopy • Investigation of signal transduction or cytoskeletal effects

ibidi MOVIE For practical details please watch our movies Performing an Angiogenesis Assay (MV 15) and Tube Formation Assay Evaluated with the Wimasis Automated Image Analysis (MV 23) on www.ibidi.com.

Fluorescence microscopy of one single strand composed of HUVEC cells. F-actin cytoskeleton stained green, and cell nuclei blue.

ibidi APPLICATION NOTES In our Application Notes Tube Formation in µ-Slide Angiogenesis (AN 19) Tube Formation in µ-Plate Angiogenesis 96 well (AN 05) Tube Formation – Data Analysis (AN 27) you will find detailed information on how to setup and optimize tube formation experiments, data analysis and interpretation.

99


µ-Slide Angiogenesis A µ-Slide used to investigate angiogenesis in tube formation assays. Also perfect for 3D cell culture and immunofluorescence staining.

Functional Cell-Based Assays

• Complete solution for tube formation experiments, requiring only a few steps from sample preparation to image analysis • Brilliant visualization without meniscus formation, and with all cells in one focal plane • Can be used with a broad range of gels, e.g. Matrigel™, collagen, and agarose* • Cost-effective experiments, requiring only 10 µl of gel per well

tube formation assay

Tube Formation Assay homogeneous tube formation cell seeding

tube formation assay

homogeneous cell seeding

tube formation

Applications • Tube formation assays • Sprouting assays • 3D cell culture

Gel matrix

Homogeneous Tube formation Gelsprouting matrix spheroid cell seeding cell spheroid

sprouting

• Immunofluorescence staining • Phase contrast microscopy of attached cells

sprouting spheroid Sprouting Spheroid cell spheroid

sprouting

Technical Features  • Standard slide format, compatible with multi-channel pipettes

sprouting aortic ring Cell spheroid Sprouting sprouting

tissue piece

sprouting aortic ring sprouting

tissue piece

• Closely fitting lid to reduce evaporation • 4 mm well within a 5 mm well • Homogeneous 0.8 mm thick gel layer* • Homogeneous cell growth • Compatible with staining and fixation

10x

• Excellent optical properties for microscopy • Made of biocompatible plastic material – no glue, no leaking

10x

*The gel matrix is not part of the product.

Cell medium (50 µl) Cells on gel 0.8 mm

“Well-in-a-well” feature avoids meniscus formation

Gel matrix (10 µl) Coverslip-like bottom 4 mm 5 mm

Specifications:

Ordering Information:

Number of wells

15

Cat. No.

Description

Volume inner well

10 µl

81506

µ-Slide Angiogenesis, ibiTreat, tissue culture treated, sterile

15

Ø inner well

4 mm

81501

µ-Slide Angiogenesis, hydrophobic, uncoated, sterile

15

Volume upper well

50 µl

81531

5 mm

For Microdissection only: µ-Slide Angiogenesis, Microdissection, PEN-membrane, sterile

15

Ø upper well

Growth area per inner well 0.125 cm² Coating area per inner well 0.23 cm2

100

Bottom: ibidi Standard Bottom

Pcs. / Box


µ-Plate Angiogenesis 96 well A µ-Plate 96 well used to investigate angiogenesis in high throughput tube formation assays

• Fully SBS (Society for Biomolecular Screening) and robotics compatible screening plate

• Cost-effective high throughput experiments, requiring only 10 µl of gel per well

Applications • Tube formation assays • Sprouting assays • 3D cell culture • Immunofluorescence staining • Cultivation and microscopy of both living and fixed cells

Technical Features: • Standard SBS format • ibidi Standard Bottom (180 µm) for high resolution microscopy • 4 mm well within a 5 mm well • Homogeneous 0.8 mm thick gel layer*

ibidi APPLICATION NOTE

• Homogeneous cell growth

The Application Note

*The gel matrix is not part of the product.

µ-Plate Angiogenesis 96 well

µ-Plate Angiogenesis 96 well

Specifications:

(180 µm)

89646

(1 mm)

Number of wells

96

Length / Width

127.7 / 85.5 mm

Height with / without lid 16.6 / 14.4 mm

µ-Plate 96 well LR Ordering Angiogenesis Information: Thick Plate Bottom Description = µ-Plate Angiogenesis 96 well, ibiTreat, tissue culture treated, Low Resolution Only sterile

Tube Formation in µ-Plate Angiogenesis 96 well (AN 05) provides practical advice on how to run tube formation assays using the µ-Plate Angiogenesis 96 well.

ibidi Standard Bottom = Coverslip-like Bottom

Cat. No.

Tube Formation & Sprouting

• Brilliant visualization without meniscus formation, and with all cells in one focal plane

Pcs. / Box 15

Well-to-well distance

9.0 mm

Volume inner well

10 µl

Ø inner well

4 mm

Volume upper well

70 µl

Ø upper well

5 mm

Growth area per inner well

0.125 cm²

Coating area per inner well

0.23 cm2

Bottom: ibidi Standard Bottom

101


WimTube – Tube Formation Image Analysis A web-based quantitative image analysis of tube formation assays for the investigation of angiogenesis

Detailed product description: page 152. • Branching points • Tube lengths, numbers, and statistics • Loop numbers, areas, and perimeters • Cell-covered area

Gel matrix

1. Cell seeding and tube formation

2. Acquisition of microscopy images

3. Quantitative image analysis

4. Data analysis and evaluation

WimSprout – Sprouting Image Analysis A web-based, quantitative image analysis of sprouting assays for the investigation of angiogenesis and tumor growth

Detailed product description: page 153.

1500 1000 500 0

C

on

Sprouting

***

1. Sprouting spheroid

2. Acquisition of microscopy images

3. Quantitative image analysis

+ VE su G bs F t. 1 + VE su G bs F t. 2

Cell spheroid

***

2000

tro l

Gel matrix

2500

VE G F

Only 4 Steps From Sample Preparation to Image Analysis

Cumulative Sprout Length in µm

Functional Cell-Based Assays

Only 4 Steps From Sample Preparation to Image Analysis

4. Data analysis and evaluation

ibidi Heating & Incubation System Live Cell Imaging with incubator conditions directly on the microscope

Migration and proliferation assays require full incubator conditions on the microscope for time lapse video microscopy. The ibidi Heating & Incubation System provides precise and reliable temperature control and gas incubation and a perfect illumination of the sample. It is compatible with all inverted microscope models.

102

Detailed product description: page 15.


Customer Feedback

Dr. Esther G.L. Koh Head, Advanced Imaging Laboratory Life Sciences Institute Immunology Programme National University of Singapore Centre for Life Sciences Singapore

„Using the „angiogenesis slide“ from ibidi is the only possibility that I know for achieving a consistently good optical quality in the tube formation assay, and saving Matrigel at the same time. In comparison to other systems, the heating stage from ibidi shows superior thermal stability and enables you to work with high humidity (>80%). This is not possible with other systems but is indispensable for longterm studies.“ Prof. Dr. Stefan Zahler Pharmaceutical Biology Munich Center for SystemBased Drug Research Ludwig-MaximiliansUniversity Germany

“I used the ibidi µ-Plate Angiogenesis 96 well for bioluminescence measurements. The plate performed exceptionally well for this specific application, as it reduced my sample volume drastically and the optical quality was just superb.”

Sam Noppen Rega Institute for Medical Research, KU Leuven Leuven Belgium

Tube Formation & Sprouting

“ibidi made it much simpler for me to prepare cells for confocal microscopy and live-cell timelapse microscopy. Cells that attached poorly to glass grew better on ibidi µ-Slides/Dishes. The angiogenesis slide allowed me to minimise the amount of cells and reagents required for an experiment.”

References Filopodia and Membrane Blebs Drive Efficient Matrix Invasion of Macrophages Transformed by the Intracellular Parasite Theileria annulata M. Ma, M and Baumgartner PLoS ONE, 2013, 10.1371/journal. pone.0075577

Tyrosine-phosphorylated Galectin-3 Protein Is Resistant to Prostate-specific Antigen (PSA) Cleavage V. Balan, P. Nangia-Makker, D. H. Kho, Y. Wang and A. Raz J. Biol. Chem., 2012, 10.1074/jbc. C111.331686

The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen C.-H. Kuo, P.-K. Chen, B.-I. Chang, M.-C. Sung, C.-S. Shi, J.S. Lee, C.-F. Chang, G.-Y. Shi and H.-L. Wu Blood, 2012, 10.1182/ blood-2011-08-376038

Heparin Strongly Induces Soluble fms-Like Tyrosine Kinase 1 Release In Vivo and In Vitro J. Searle, M. Mockel, S. Gwosc, S. A. Datwyler, F. Qadri, G. I. Albert, F. Holert, A. Isbruch, L. Klug and D. N. Muller Arteriosclerosis, Thrombosis, and Vascular Biology, 2011, 10.1161 ATVBAHA 111.237784

Diosgenin, a Steroidal Saponin, Inhibits Migration and Invasion of Human Prostate Cancer PC-3 Cells by Reducing Matrix Metalloproteinases Expression P. S. Chen, Y. W. Shih, H. C. Huang and H. W. Cheng PLoS ONE, 2011, 10.1371/journal. pone.0020164

103


Experimental Workflow

Functional Cell-Based Assays

Chemotaxis 0 min

10 min

20 min

Sample Preparation

Video Microscopy

Cell Tracking

µ-Slides for Chemotaxis (page 109)

Microscope with Incubation (page 15)

Manual or Automated Cell Tracking (page 154)

µ-Slide Chemotaxis 2D The µ-Slide Chemotaxis 2D is optimized to analyze the chemotactical response of adherent cells in linear and stable concentration profiles. Due to the gradient’s time stability of over 48 hours, it is the first system that is able to analyze the chemotaxis of slow migrating cells for up to two days. For use with strongly adherent cells only, such as: endothelial cells, fibroblasts, cancer cells, and others.

Principle: A narrow observation area connects two large reservoirs. The cells that are seeded into the observation area become super-imposed by linear and time-stable gradients. Chemotaxis is observed using time-lapse microscopy. The ibidi Heating & Incubation Systems (see page 15) enable live cell imaging with incubator conditions directly on the microscope.

Observation area is recorded with a 4 x objective.

104

Chemotaxis of HT-1080 cells, which are migrating towards FCS, using low resolution microscopy for a maximum of migration data

High resolution microscopy during chemotaxis and cell polarization, using the HT-1080 LifeAct cell line (see page 34)


10 10 10

-4 0.3 -8

FMI

p-value (Rayleigh-Test)

10 0

-12

+/-

-/-

0.2 0.1 0.0

+/+

+/-

Data Table

Visual Analysis

-/-

+/+

Quantitative and Statistical Analysis

Wimasis WimTaxis and/or ibidi Chemotaxis and Migration Tool (page 155)

Chemotaxis Chemotaxis

µ-Slide Chemotaxis 3D The µ-Slide Chemotaxis 3D was developed to investigate the chemotactical behavior of fast or slow migrating, non-adherent cells in gel matrices. It is possible to observe the migration in linear and stable concentration profiles for over 48 hours. As gradients are rapidly established, fast responses (occurring in less than 30 minutes) can also be measured. For use with all adherent cells that will be observed in 2D or in in vivo-like 3D gel conditions, such as endothelial cells, fibroblasts, cancer cells, and others and non-adherent cells, such as T-cells, dendritic cells, neutrophils, monocytes, granulocytes, lymphocytes, and others.

Principle: A narrow observation area connects two large reservoirs. The cells that are embedded in a gel inside the observation area become super-imposed by linear and time-stable gradients. Chemotaxis is observed using time-lapse microscopy.

Observation area is recorded with a 4 x objective.

HT-1080 cells migrating towards FCS inside a gel matrix (objective lens 10 x).

Dendritic cell (mouse) with LifeActlabeled F-actin that is migrating towards CCL 19 inside a Collagen I gel matrix. Spinning disc confocal microscopy (objective lens 63 x).

105


µ-Slide Chemotaxis 3D

µ-Slide Chemotaxis 2D 100

Concenctration

Observation Area

Observation Area

01 h 04 h

75

12 h 48 h 50

25

50 0

10 00

0

-5 00

The ibidi µ-Slides for chemotaxis are designed to provide excellent long-term gradient stability for over 48 hours. They are especially made for working with slow migrating (cancer) cells with data from at least 12 hours, in which the cells migrated a significant distance.

Distance from Center [µm]

Gradient measurements across the observation area in µ-Slide Chemotaxis 2D and µ-Slide Chemotaxis 3D

2D Versus 3D It is commonly accepted that cells in a culture behave very differently when they are attached to a flat 2D surface, compared to being inside a 3D gel matrix. In most cases, the 3D environment mimics the in vivo situation much better. Gradients for chemotaxis can easily be built up in water-based gels (like Collagen I gels, see page 52), because the gel structure does not hinder diffusion.

Example of HT-1080 cancer cells on a 2D surface (top), and embedded into a Collagen I gel (bottom)

2D Migration

3D Migration

Focal adhesions α5β3 integrin receptor mediated

Migration in ECM-like matrix

Always mesenchymal

Ameboid and / or mesenchymal

Easy comparable results

Results strongly dependent on the gel that is used

Easy to perform assay

More complex assay

0.3

FMI

Functional Cell-Based Assays

Distance from Center [µm]

-1 00 0

50 0

10 00

0

-5 00

-1 00 0

0

Gradient Stability

0.2

Statistics

0.1 0.0

+ /-

-/-

+/+

Typical results of a chemotaxis experiment with HT-1080 cells migrating towards fetal calf serum. The FMI (forward migration index) values parallel (II) and perpendicular (  _I ) to the gradient are shown. Significant chemotaxis is only observable parallel to the gradient in the +/- experiment. All other values are not significantly different from 0.

Typically, three to five repeat experiments are sufficient to create significant data from chemotaxis and control experiments. To achieve this, each experiment should contain data from 20 - 40 single cells, which is possible using low-magnification microscopy objective lenses, such as 5 x or 10 x.

Experimental Endpoints • Chemotactic behavior of cells and mutants (knockout / knockdown) • Chemotactic potential of substances • Effect of chemotaxis inhibitors / enhancers • Distinguishing between chemotaxis and chemokinesis (enhanced migration) • Molecular mechanisms during chemotaxis, visualized by fluorescence microscopy

106

• Investigation of signal transduction or cytoskeletal effects during chemotaxis


Experimental Examples

+

+

-

-

HUVEC Chemotaxis Inhibition Endothelial cells, which tend to migrate in a collective manner, can be tested on compounds that inhibit chemotaxis functionality, even when there is a gradient present (e.g., compounds suppressing cytoskeleton dynamics, or receptor antagonists).

Without inhibitor – Chemotaxis

With inhibitor – No chemotaxis

Leukocytes

Cancer Cells Chemotaxis, described as the directed motion towards a chemoattractant, can be distinguished from chemokinesis, which is known as an enhanced migration effect. Both effects can be analyzed independently of each other. In the example, the chosen chemoattractant induces both chemotaxis and chemokinesis.

+

+

-

-

+

-

Chemotaxis

Enhanced migration (chemokinesis)

Reduced migration (chemokinesis)

Cell type 1 in reservoir producing chemoattractant

Cell type 2 in gel matrix doing chemotaxis

Chemotaxis Chemotaxis

Fast leukocytes, such as dendritic cells or T-cells, can be visualized well during chemotaxis, when inside gel matrices like collagen. This is especially true when using high-resolution fluorescence microscopy, which shows changes in the cytoskeleton during immune response and 3D interstitial migration.

Cell-to-Cell Chemotaxis The ibidi µ-Slide Chemotaxis 3D (see page 109) can additionally be used for various cell-to-cell chemotaxis assays. For example, cells brought inside the large reservoirs can be used as a chemoattractant producer.

ibidi MOVIE For practical details please watch our movies ibidi Chemotaxis Assays (MV 25) Chemotaxis of HT-1080 in µ-Slide Chemotaxis 2D (MV 08) Chemotaxis of HUVEC Blocked by Spongistatin (MV 09) on www.ibidi.com.

ibidi APPLICATION NOTE In our Application Notes 14, 17, and 23 you will find detailed information on how to perform your chemotaxis experiments: Chemotaxis 2D (AN 14) Chemotaxis 3D (AN 17) 3D Chemotaxis Protocol for Non-Adherent Cells in a Gel Matrix (AN 23)

107


µ-Slide Chemotaxis Family Used to investigate chemotaxis of fast or slow migrating adherent or non-adherent cells

• Chemotaxis measurement in real-time

Functional Cell-Based Assays

• Stable gradients for long-term experiments • Easy handling and analysis • Reproducible results with reliable and user-independent data

Applications • Chemotaxis of adherent cells on 2D surfaces, e.g., cancer cells, endothelial cells, and fibroblasts

ibidi APPLICATION NOTE

• Chemotaxis of adherent or non-adherent cells in 3D gel matrices

The Application Notes Chemotaxis 2D (AN 14) and Chemotaxis 3D (AN 17) provide practical advice on how to perform your chemotaxis 2D and 3D experiments.

• 3D chemotaxis of leukocytes or cancer cells in ECM-like matrix • Invasion assays of tumor cells in Matrigel™

Technical Features • 3 chambers on one slide for parallel assays • Ready to use, i.e., no assembly required • Linear gradients, which remain stable for over 48 hours • Made for high-end video microscopy, including fluorescence microscopy • µ-Slide Chemotaxis 3D ideal for collagen gels, hydrogels, Matrigel™, or similar aqueous gels Preparation

Seed cells with gel matrix

C0 C100

Fill with chemoattractant-free medium

Fill with chemoattractant

Basic Principle Two large reservoirs of 60 µl are connected by a narrow observation area. The adherent cells or the cells embedded in a gel inside the observation area become super-imposed by a linear and time-stable gradient.

Chemotaxis on a 2D Surface

C100 Cells on 2D surface

108

1 mm

Chemotaxis in a 3D Gel Matrix

C0

C100

70 µm

Cells in 3D matrix

1 mm

C0 70 µm


µ-Slide Chemotaxis 3D Used to investigate chemotaxis of fast or slow migrating adherent cells and non-adherent cells in gel matrices

Observation Area of the µ-Slide Chemotaxis 3D in 2D and 3D Chemotaxis Assays

Cells on 2D surface

Ordering Information:

ibidi TIP

Cat. No.

Description

Pcs. / Box

80326

µ-Slide Chemotaxis 3D, ibiTreat, tissue culture treated, sterile

10

80322

µ-Slide Chemotaxis 3D, Collagen IV, sterile

10

The µ-Slide Chemotaxis 3D is recommended for 2D and 3D applications.

Chemotaxis

Cells in 3D matrix

Specifications: Chemotaxis chambers on slide 3

Distance between chambers 18.5 mm

Volume per chamber

120 µl

Total height with plugs

12 mm

Observation area

2x1 mm²

Volume chemoattractant

30 µl

Coating area per chamber - When coating full chamber - When coating observation area only

3.50 cm2 0.27 cm2

Bottom: ibidi Standard Bottom

µ-Slide Chemotaxis 2D Used to investigate chemotaxis of slow migrating adherent cells on 2D surfaces

Ordering Information: Cat. No.

Description

80306

µ-Slide Chemotaxis 2D, ibiTreat, tissue culture treated, sterile

Pcs. / Box 10

80302

µ-Slide Chemotaxis 2D, Collagen IV, sterile

10

Specifications: Chemotaxis chambers on slide 3

Distance between chambers

21.5 mm

Volume per chamber

80 µl

Total height with plugs

12 mm

Observation area

2x1 mm2

Volume chemoattractant

18 µl

Coating area per chamber - When coating full chamber - When coating observation area only

2.40 cm2 0.39 cm2

Bottom: ibidi Standard Bottom

NOTE: The µ-Slide Chemotaxis 2D is for use only with strongly adherent cells.

109


sticky-Slide Chemotaxis 3D A bottomless slide used in 2D and 3D chemotaxis applications with a self-adhesive underside designed to allow self-assemblage of the slide setup

Functional Cell-Based Assays

• Versatile – i.e., permits the mounting of a variety of bottom materials • Allows chemotaxis measurements to be taken in real-time • Provides stable gradients for long-term experiments

Applications • Inserting materials or tissue into chemotaxis chambers • Keeping cell spheroids or tissue in a stable concentration gradient • Chemotaxis of fast or slow migrating cells on either a 2D surface or in 3D gels • Usable with a range of bottom materials, such as plastic sheets, silicon chips, and glass slides

Technical Features • Bottomless slide

Sample

sticky-Slide with channel

Sample preparation

• Self-adhesive underside • Biocompatible adhesive that has been cell culture tested • Adheres to all flat surfaces, even wet surfaces

Coverslip

• Suitable coverslips available Pressing and sample fixation

Assembled chemotaxis slide

• Specifications identical to those of the µ-Slide Chemotaxis 3D (see page 109), except that it has no bottom

Removing the protection film Exposed selfadhesive underside

Chemotaxis experiment

Specifications:

110

Assembling with substrate

Ordering Information:

Chemotaxis chambers on slide

3

Volume per chamber

120 µl

Observation area

2x1 mm²

Total height with plugs

12 mm

Volume chemoattractant

30 µl

Bottom

none

Cat. No.

Description

80328

sticky-Slide Chemotaxis 3D, sterile

10812

Coverslips for sticky-Slides, D 263 M Schott glass, No. 1.5H (170 µm +/- 5 µm), unsterile, 25 mm x 75 mm,

Pcs. / Box 10 100


µ-Slide III 3in1 A µ-Slide for switchable chemotatic gradients that merges 3 separated liquids into one channel

• Dynamic fluidic gradients with a maximum time resolution • Alternating gradient fields for chemotactic cell trapping

Applications • Fluidic assays with up to three different liquids • Cell sorting with laser traps • Fluidic focusing of inner lane

Chemotaxis

Technical Features • 3 channels ending in 1 • Microfluidic bi-directional gradient generator • Fully compatible with the ibidi Pump System (see page 118) and other pumps • The flow is always laminar • Contains a millimeter scale bar Flow Setup for Switchable Chemotactic Gradients Flow direction 5

10

15

20

*

Concentration [a.u.]

0

0.4 0.3 0.2

State 1

0.1

State 2

0.0 0

1

2

3

Channel width at * position [mm]

Gradient steepness at different positions. The flow is generated by the ibidi Pump System (see page 118).

Overlapping gradients for chemotactical cell trapping. Switching times of some seconds allow fast gradient switching, so that cytoskeletal dynamics can be observed using fluorescence microscopy.

Ordering Information: Cat. No. 80316

Description µ-Slide III 3in1, ibiTreat, tissue culture treated, sterile

Specifications: Pcs. / Box 15

80312

µ-Slide III 3in1, Collagen IV, sterile

15

80313

µ-Slide III 3in1, Fibronectin, sterile*

15

80314

µ-Slide III 3in1, Poly-L-Lysine, sterile

15

80315

µ-Slide III 3in1, Poly-D-Lysine, sterile*

15

80311

µ-Slide III 3in1, hydrophobic, uncoated, sterile

15

* available only on request

Adapters

Female Luer

Volume per reservoir

60 µl

Number of channels

3 in 1

Total channel volume

60 µl

Height of all channels

0.4 mm

Width of channels thin/thick 1 / 3 mm Total growth area

1.23 cm²

Coating area

3.05 cm2

Distance of scale bars

1 mm

Bottom: ibidi Standard Bottom

111


WimTaxis – Chemotaxis Image Analysis A web-based, quantitative image analysis of chemotaxis assays with automated tracking of label-free cell lines and bacteria in phase contrast

Only 4 Steps From Sample Preparation to Image Analysis

Detailed product description: page 154.

10 0 10 10 10

-4 -8

100

0

0.3

-100 FMI

20 min

y displacement [µm]

10 min

p-value (Rayleigh-Test)

Functional Cell-Based Assays

200

0 min

-12

-200 +/-

-/-

-200

0.2 0.1 0.0

+/+

-100

+/-

0

-/-

100 +/+

200

x displacement [µm]

1. Cell seeding and chemotaxis assay

2. Acquisition of microscopy images

3. Automated cell tracking

4. Data analysis and evaluation

Chemotaxis and Migration Tool A free software tool for data analysis from time stack chemotaxis experiments, based on the National Institute of Health’s (NIH) ImageJ image processing system

• A free and platform independent solution • Provides various graphs and statistical tests that allow advanced analysis of chemotaxis experiments The Chemotaxis and Migration Tool is freely available on www.ibidi. com. The tool provides various graphs and statistical tests that allow advanced analysis of experimental data. For a detailed product description please refer to page 155.

Collagen Type I N EW

A high quality, rat tail collagen solution used for creating 3D collagen gels (e.g., in chemotaxis assays)

• Provides in vivo–like ECM (extracellular matrix) structures • High concentrated collagen solution – usable for a variety of gel concentrations • Fast polymerizing for optimal cell distribution in gel • Non-pepsinized, native collagen for a close-to-nature situation

112

For a detailed product description please refer to page 52.


Customer Feedback

“The ibidi µ-Slides Chemotaxis 2D and 3D have been the key to my research on lymphocyte chemotaxis. These slides have allowed me to track and visualize directional motility in lymphocytes. The generated chemokine gradients are stable for over 24 hours and the viability of the cells is amazingly good, allowing for measurements of chemotaxis over prolonged time frames.

PD Dr. Peter Hanley University of Münster Institute of Molecular Cell Biology Münster Germany

Loïc Dupré PhD INSERM UMR 1043 Purpan University Hospital Toulouse France

Chemotaxis

I am also using the ibidi Gas Incubation and Temperature Control Systems, which are robust and guarantee perfect stability. Very importantly, the product developers at ibidi provide a great support for the implementation of their systems. Thanks to ibidi for developing cell imaging products of such great quality!”

“In the past 5 years, we have extensively used the ibidi µ-Slide Chemotaxis 2D to perform time-lapse images of primary mouse macrophages migrating in a chemoattractant gradient. We are most grateful that this device was developed, otherwise it would have been technically difficult to perform robust chemotaxis assays over long time periods (in our case, up to 14 h).”

References The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration P. Suraneni, B. Rubinstein, J. R. Unruh, M. Durnin, D. Hanein and R. Li The Journal of Cell Biology, 2012, 10.1083/jcb.201112113 Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy S. H. Ngalim, A. Magenau, Y. Zhu, L. Tønnesen, Z. Fairjones, J. J. Gooding, T. Böcking and K. Gaus Journal of Visualized Experiments, 2012, doi:10.3791/50310

DOCK8 is a Cdc42 activator critical for interstitial dendritic cell migration during immune responses Y. Harada, Y. Tanaka, M. Terasawa, M. Pieczyk, K. Habiro, T. Katakai, K. Hanawa-Suetsugu, M. KukimotoNiino, T. Nishizaki, M. Shirouzu, X. Duan, T. Uruno, A. Nishikimi, F. Sanematsu, S. Yokoyama, J. V. Stein, T. Kinashi and Y. Fukui Blood, 2012, 10.1182/ blood-2012-01-407098 Sphingosine 1-PhosphateInduced Motility and Endocytosis of Dendritic Cells Is Regulated by SWAP-70 through RhoA C. Ocana-Morgner, P. Reichardt, M. Chopin, S. Braungart, C. Wahren, M. Gunzer and R. Jessberger The Journal of Immunology, 2011, 10.4049/jimmunol.1003461

Investigation of the marine compound spongistatin 1 links the inhibition of PKCα translocation to nonmitotic effects of tubulin antagonism in angiogenesis A. S. Rothmeier, I. Ischenko, J. Joore, D. Garczarczyk, R. Fürst, C. J. Bruns, A. M. Vollmar and S. Zahler FASEB J , 2008, 10.1096/ fj.08-117127.

113


Cell Culture Under Shear Stress

Functional Cell-Based Assays

In vivo, several adherent cell types are exposed to mechanical shear stress in biofluidic systems such as blood or lymphatic vessels and nephrons. This mechanical stimulus has a great impact on the physiological behavior and adhesion properties of cells.

Shear stress

No shear stress

For more convenient examination of perfusion assays, ibidi has developed a special pump system (i.e., the ibidi Pump System, see page 118) and special flow chambers for use in shear stress experiments, i.e., the µ-Slide I Luer Family (see page 128), µ-Slide VI 0.4 (see page 131) and µ-Slide VI 0.1 (see page 130).

Shear Stress, in General Static

Perfusion

Human Vessel

Shear stress (dyn / cm2) Average

Maximal

Aorta

5

> 15

Artery

5 - 10

20 - 70

Middle vein

0.8 - 1

-

0.5

-

Small vein

Unidirectional Laminar Flow

... is the mechanical force induced by the friction of liquid against the distal cell membrane. Cells are able to countervail deformations caused by shear stress by rearranging their cytoskeleton and the cell-cell contacts. Other shear stress dependent effects include changes in metabolism, gene expression, and differentiation. Physiological shear stress values vary from 0.5 to 120 dyn / cm2 depending on the vessel type (e.g., artery or vein) and the size of the organism (e.g., mouse, rat, or human).

Unidirectional Laminar Flow

Flow rate

1.0 0.5 0.0 -0.5 -1.0

Time

Area of homogeneous shear stress

...  is encountered in most small healthy biological vessels, such as small arteries and veins. Certain cells, such as endothelial cells and kidney epithelial cells, are in vivo constantly exposed to flow. Experimentally, this is achieved by perfusing medium through low walled channels, and by keeping the flow constant over time for both direction and velocity. Homogeneous laminar shear stress covers the whole channel area of the slide, except for a thin band on either side of the channel walls and near the reservoirs. The width of these bands is proportional to the channel’s height.

Recommended observation area

Pulsatile Laminar Flow

For homogeneous laminar flow experiments the µ-Slide I Luer Family and the µ-Slide VI 0.4 are recommended.

Pulsatile Laminar Flow

Flow rate

1.0 0.5 0.0 -0.5 -1.0

Time

114

... is encountered in arterial vessels due to the fluctuations caused by the heartbeat. Experimentally, this type of flow can be mimicked by employing unidirectional flow with a periodically changing flow rate while keeping the flow direction constant.


Turbulent Flow

Turbulent Flow 0.5 0.0 -0.5 -1.0

Time

NOTE: Due to physical reasons, turbulent flow cannot be achieved in ibidi flow chambers using physiological flow regimes.

Oscillating Flow Flow rate

… is accepted as a means of simulating turbulences when using flow chambers. Although the flow is laminar, there is no main direction due to the fact that the direction of the flow is changed at regular intervals (often this interval is every 0.5 s). Other than during valve switching, the flow rate is kept constant.

Oscillating Flow 1.0 0.5 0.0 -0.5 -1.0

Time

100 %

Excurse: Non-Uniform Laminar Shear Stress ... occurs in vivo at branching sites and at other regions of disturbed blood flow in vessels. Experimentally, nonuniform laminar shear stress can be achieved by spatially varying flow rates. It can be used for investigating cells at different shear stresses using a single sample. In more complex experiments, it can be used for studying cells and cellular signaling when the cells are exposed to areas of strongly varying shear stress. The µ-Slide y-shaped (see page 132) was designed to help conduct studies of non-uniform shear stress. In the branched region of the slide, the prevalent shear stress is approximately half of that found in the single straight channel regions. Researchers should refer to Application Note 18 for numerical simulations of exact shear stress values at the branching points. Please note that it is experimentally impossible to study turbulences in a µ-Slide y-shaped.

Live Cell Perfusion Imaging & Shear Chemotaxis Stress

... near surfaces is characterized by changes in flow rate and direction. Direction and velocity change over time, thus the flow profile is not constant. In vivo, turbulences are rare and can only be found during pathophysiological processes. In many cases, turbulences are falsely considered to appear around plaque that has developed at the largest branching of the Arteria Carotis.

Flow rate

1.0

0%

ibidi APPLICATION NOTE AN 11 Shear Stress and Shear Rates provides detailed information on shear stress / shear rates and flow rates in ibidi’s channel slides.

Influence of Shear Stress on Cultured Cells Human umbilical vein endothelial cells (HUVEC) cultured under flow conditions (20 dyn / cm²) in a µ-Slide I 0.4 Luer over 9 days. The primary cells were transduced with the adenoviral vector rAV CMV-LifeAct-TagRFP (see page 33) 24 hours prior to the experiment.

115


Types of Assays

Functional Cell-Based Assays

Cell Culture under Shear Stress

Rolling and Adhesion Assays

Application:

• Investigating the influence of shear stress on endothelial cells • Preparing cells while mimicking in vivo perfusion conditions • Investigating platelet or lymphocyte adhesion to endothelial cells cultured under in vivo-like conditions • Creating biofilms of microorganisms • Staining antibodies Flow Characteristics:

Continuous laminar, non-uniform, oscillating (needed for turbulence simulation), pulsatile

Duration:

Hours, up to several weeks

Experimental Environment:

Incubation conditions

Recommended Pumps:

ibidi Pump System

Recommended µ-Slides:

µ-Slide VI, µ-Slide I Luer Family, µ-Slide III

Application:

Rolling and adhesion of suspended cells to protein surfaces: • Blood cells (e.g., platelets, leukocytes, and monocytes) on adhesion proteins • Blood cells on confluent cell monolayers

Stop Flow Experiments

3D Cell Culture: Interstitial Flow

Flow Characteristics:

Continuous laminar

Duration:

Minutes to hours

Experimental Environment:

Room temperature or incubation conditions

Recommended Pumps:

ibidi Pump System, Syringe pumps

Recommended µ-Slides:

µ-Slide VI, µ-Slide I Luer Family, µ-Slide y-shaped

Application:

Defined liquid exchange: • Ca2+ -imaging • Defined medium exchange for optimal feeding

• Online drug delivery • Live staining

Flow Characteristics:

Laminar flow with short periods of flow in between non-flow phases

Duration:

Minutes to hours

Experimental Environment:

Room temperature or incubation conditions

Recommended Pumps:

Manual liquid delivery, Syringe pumps, Peristaltic pump

Recommended µ-Slides:

µ-Slide VI, µ-Slide I Luer Family, µ-Slide I

Application:

3D cultures of cells and tissue in gel matrices: • Hepatocytes • Fibroblasts • Muscle cells

Cells in 3D culture

• Kidney cells • Stem cells

Flow Characteristics:

Continuous interstitial or stop flow interstitial

Nutrients

Duration:

Hours, up to several weeks

O2, CO2

Experimental Environment:

Incubation conditions

Recommended Pumps:

ibidi Pump System

Recommended µ-Slides:

µ-Slide VI, µ-Slide I Luer Family

Gel matrix

116

Endothelial cells under flow:


ECIS Flow Assays ECIS technology permits the measurement of even small morphological changes in electrical signals, without disturbance to the cells, and without requiring any staining. The measurements are automated and are therefore highly reproducible with minimum effort.

Suitability of Various Channel Heights for Flow or Longer Term Static Cultivation Channel height

0.1 mm

0.2 mm

0.4 mm

0.6 mm

0.8 mm

µ-Slides

Static cultivation for long time Perfusion Experiments

How to Identify the Ideal Pump for Your Experiment

Live Cell Perfusion Imaging & Shear Chemotaxis Stress

The ECIS Flow Array (see page 146) enables researchers to investigate cells under both perfusion and defined shear stress conditions.

Depending on experimental requirements, different pump solutions can be used, as outlined in the table below: Syringe Pump (see page 126)

Peristaltic Pump

ibidi Pump System (see page 118)

Almost none (only initial pulse)

Pulsation via drive shaft

Almost none (only during valve activation)

Possible, but only with push-and-pull pumps

Unlimited

Unlimited

Possible, but volume is limited

Possible, but volume is limited

Possible

Pulsatile flow for simulating heart beats

Not possible

Not possible

Included in software

Oscillating flow for simulating turbulences

Not possible

Not possible

Included in software

Almost none, but sedimentation can be a problem

Strong

Almost none

Excellent

Difficult due to pulsation

Excellent

Flow velocity

Volume is limited

Unlimited

Unlimited

Parallelization

Possible

Possible

Possible

Ease of set up within an incubator

Difficult

Difficult

Easy

Limiting factor

Small

Small

Yes

Yes

Yes

Pulsation Circulating flow Unidirectional flow

Mechanical stress during pumping on non-attached cells held in a reservoir Combination with microscopy

Required volume Programmable

117


ibidi Pump System Ideal simulation of physiological flow conditions

Functional Cell-Based Assays

ibidi Pump

The ibidi Pump System enables the cultivation of endothelial cells under perfusion to reflect their natural environment far better than by doing so under static conditions.

• Creates a continuous unidirectional, oscillating, or pulsatile flow • Operates up to four parallel Fluidic Units

Fluidic Unit • Works with minimal amounts of medium and supplement • Also available as a Fluidic Unit Quad for parallel flow assays in four µ-Slides

PumpControl Software

Applications

• Easy setup of an automated flow control for flow assays

• Defined shear stress in long-term cell culture (e.g., endothelium, kidney, or biofilm)

• Internal calculation for flow rates, shear rates, and shear stress

• Live cell imaging and immunofluorescence for analyzing shear stress response • Mimics shear stress conditions in microcapillary, venous, and arterial flow • Rolling and adhesion of blood cells to protein surfaces

Experimental Examples:

rAV CMV-LifeAct-TagRFP-transduced HUVEC, cultivated under 20 dyn / cm² in µ-Slide I 0.4 Luer

118

HUVEC, cultivated over 7 days at 10 dyn / cm². VE-cadherins are stained in green, cell nuclei are stained in blue.

Rolling and adhesion of polymorphonuclear leukocytes on LPS-stimulated cerebrovascular endothelial cells in µ-Slide VI 0.4 at 1 dyn / cm². (Courtesy of G. Cepinskas, Ontario)


ibidi Pump System A pump system for the cultivation of cells under flow for simulation of blood vessels

ibidi Pump

+ Fluidic Unit

PumpControl

Optional: Fluidic Unit Quad

• Ideal simulation of various physiological conditions – continuous unidirectional, oscillating, and pulsatile flow • Suitable for long-term live cell imaging • Compatible with the ibidi Heating Systems, all incubators, and incubated microscopes • Minimal amount of medium and supplement needed

ibidi TIP Contact ibidi for a free demo of the ibidi Pump System.

Components of the ibidi Pump System PumpControl Software

ibidi Pump • Computer-controlled air pressure pump • Operates up to four parallel Fluidic Units per ibidi Pump

• Easy setup of an automated flow control for flow assays • Internal calculation for flow rates, shear rates, and shear stress

N

Fluidic Unit

Perfusion & Shear Stress

+

Fluidic Unit Quad

EW

• A holder for one Perfusion Set (10 ml) and connected μ-Slide(s) • For placement in an incubator • Optional: Reservoir Holder for 2 ml and 50 ml Perfusion Sets

Perfusion Set • Includes tubing, adapters, and reservoirs to perform flow assays • Designed for various flow rates and volumes

• Combines four Fluidic Units into one system • Operated by a single ibidi Pump

Heating Insert Adapter for Perfusion Assay • Adapter for the ibidi Heating System (see page 19) • For long-term cell studies (several days) under flow conditions

119


Experimental Workflow Flow Conditioning of Adherent Cells

Staining and Image Acquisition

Principle of Flow Generation

Ambient air Ambient air

CO2 Incubator

Air flow

CO2 Incubator

Applying pressured air to the reservoirs of the Fluidic Unit generates the flow in the µ-Slide channels. In order to avoid wasting any medium, the liquid is pumped back and forth from reservoir A to reservoir B by switching the valve V1.

Ambient air Ambient air

on V1

off

A

V1

Reservoirs

In state 1, the pressured air is guided to reservoir A while reservoir B is connected to the ambient air pressure. In state 2, it is the other way around. Switching between these states creates a flow of medium between the reservoirs and ensures that they do not run dry. However, to generate a unidirectional flow, a fluidic rectifier is placed between the reservoirs and the µ-Slide. The rectifier is a pinch valve V2 that clamps off two branches of the Perfusion Set while the others are kept open. The synchronous switching of both valves creates a continuous and unidirectional flow without wasting any medium.

Quantitative and Statistical Analysis

Air pressure pump (positive pressure)

Reservoirs

Functional Cell-Based Assays

Cell Seeding Into Channel Slides

B

A

B

Liquid flow on off

Technical Features

V2

V2

Marking

Marking

µ-Slide

µ-Slide

State 1

State 2

System Overview:

• Up to four parallel Fluidic Units per ibidi Pump • Flow characteristics: unidirectional and continuous flow, oscillating flow for simulating turbulent flow, and pulsatile flow

Air tubing

Incubator

• Flow rate: 0.03 – 35 ml / min • Shear stress: 0.3 – 150 dyn / cm2 • Working volume: 2.5 / 12 / 50 ml

Drying bottle Pump

• Suitable for all µ-Slides with Luer adapters

Electrical connection

• Also suitable for home-made flow chambers • Compatible with all incubators • Software-controlled flow rates and shear stress

120

Computer

µ-Slide


The ibidi Pump System Generates Minimal Mechanical Stress

Perfusion Set-Up on a Microscope

Activation of suspended dendritic cells after 24 h 100

Activation [%]

80

60

40

20

Static control (Petri Dish)

ibidi Pump System

Peristaltic pump

Chemical activation (LPS)

Ideal in combination with the ibidi Heating & Incubation System (see page 15)

ibidi MOVIE Watch our movie Long-Term Cell Culture Under Perfusion (MV 21) on www.ibidi.com to get more information on applications and handling.

Ordering Information: ibidi Pump System Cat. No.

Description

Pcs. / Box

10902

ibidi Pump System: ibidi Pump, Fluidic Unit, Perfusion Set, notebook, PumpControl software

1

10905

ibidi Pump, accuracy: +/- 1 mbar, pressure range: -100 to +100 mbar, control for up to 4 Fluidic Units, including Pump Control software

1

10903

Fluidic Unit, switching valves for various flow assays, suitable for all Perfusion Sets and channel µ-Slides, stable aluminum housing, exchangeable reservoir holders

1

10904

Fluidic Unit Quad: 4 Fluidic Units on a stable plate, switching valves for various flow assays, suitable for all Perfusion Sets and channel µ-Slides, stable aluminum housing

10908

Notebook, ready to use, pre-configured Windows system, PumpControl software

1

10991

Cell culture incubator, small capacity system (14l), specially suited for setups employing up to 2 Fluidic Units, or for separated setups like ECIS measurements

1

Perfusion & Shear Stress

0

Accessories for the ibidi Pump System Cat. No.

Description

10961

Perfusion Set BLUE, length 15 cm, ID 0.8 mm, 10 ml reservoirs, designed for low flow rates.

3

10962

Perfusion Set RED, length 15 cm, ID 1.6 mm, 10 ml reservoirs, designed for high flow rates.

3

10963

Perfusion Set WHITE, length 50 cm, ID 0.8 mm, 10 ml reservoirs, designed for low flow rates and microscopy.

3

10964

Perfusion Set YELLOW-and-GREEN, length 50 cm, ID 1.6 mm, 10 ml reservoirs, designed for high flow rates and microscopy.

3

10965

Perfusion Set YELLOW, length 15 cm, ID 0.5 mm, 2 ml reservoirs, designed for low volumes and µ-Slides with 0.1 mm channel height.

3

10966

Perfusion Set BLACK, length 50 cm, ID 0.5 mm, 2 ml reservoirs, designed for low volumes, µ-Slides with 0.1 mm channel height, and microscopy.

3

10971

Filter / Reservoir Set, 10 ml: 2 replacement reservoirs with mounted filters, ready to use, sterile

10

10972

Filter / Reservoir Set, 2 ml: 2 replacement reservoirs with mounted filters, ready to use, sterile

10

10974

Filter / Reservoir Set, 50 ml: 2 replacement reservoirs with mounted filters, ready to use, sterile

10

10976

Reservoir Holder for Fluidic Unit, 10 ml

1

10977

Reservoir Holder for Fluidic Unit, 2 ml

1

10978

Reservoir Holder for Fluidic Unit, 50 ml

1

The Perfusion Sets have ready-to-use tubing for flow assays using the ibidi Pump System. Tubes and connectors are sterile and autoclavable. The sets consist of two reservoirs and reusable silicone tubes.

Pcs. / Box

121


Impedance Measurements Under Flow Stimulation

Functional Cell-Based Assays

In vivo, endothelial cells develop and differentiate under shear stress conditions. When starting cell-based assays that use endothelial cells, you should consider the possible influence of this mechanical force on cell morphology and physiology. There is growing evidence that under in vitro conditions, the mechanical perturbations have a profound effect on the characteristics of the cell layer. In long-term experiments with HUVEC, three different phenotypes can be observed: a round flat cell after seeding, elongated cells after 1-3 days, and finally a cobblestone appearance of a dense compact cell layer. The morphological changes are subsequently accompanied by physiological changes of the endothelial cell monolayer, which is measured by impedance monitoring. Cell layer

Working Principle of Impedance Measurements

Membrane capacitance

Cells are cultivated in channels with electrode arrays. Depending on the morphological appearance of the endothelial cell monolayer, the gaps between the cells (influenced by the cell-cell contacts) change in size. These shifting gaps can be characterized by their change in conductivity when applying AC currents of different frequencies. The continuous red lines in the scheme represent the ion currents between the cells when applying the AC current.

Electrode

Barrier function

For a detailed description, please refer to the chapter “ImpedanceBased Cell Assays� (see page 137).

Experimental Example: Conductivity and Impedance Change of an Endothelial Cell Monolayer

Electrode with HUVEC under static conditions 101

101

1

2

3

4

5

6

7

8

9

Time (days)

Static culture: After seeding, cells grow to confluence in 2 - 3 days. During this time, the conductivity is reduced, and the resistance rises to a plateau, which is then maintained under the subsequent static culturing over several days.

122

flow

Norm. Impedance

static

Norm. Impedance 100

Electrode with HUVEC under shear stress conditions

100

1

2

3

4

5

6

7

8

9

Time (days)

Flow culture: Applying shear stress reduces the conductivity and increases the impedance of the monolayer. Over a period of days the impedance of the endothelial monolayer decreases. The physiological properties of the cell monolayer are altered when compared to the static conditions.


Immunofluorescence Staining of FlowConditioned Endothelial Cells

Perfusion Set-Up in the Incubator

Immunofluorescence stainings can easily be done in ibidi channel µ-Slides, subsequent to the performance of your specific experiment. When comparing the cultivation of HUVEC under static and flow conditions, the differences between static culture and flow conditioning are clearly visible. For detailed information about immunofluorescence staining in ibidi µ-Slides and µ-Dishes, please refer to page 74.

Adherence Junctions (VE-Cadherins) Flow-conditioned cells are elongated and show distinct stress fibers, whereas static culturing generates cells with a bigger surface and a chaotically structured actin skeleton. VECadherins (adherence junction proteins of endothelial cells) are present in both conditions. HUVEC, flow-conditioned, 10 dyn/cm², 5 days µ-Slide I 0.4 Luer, ibiTreat

HUVEC, static culture, 0 dyn/cm2, 5 days, µ-Dish 35 mm, ibiTreat

Red – F-Actin (Phalloidin-Alexa 633), Green – VE-Cadherin (VE-Cadherin (D87F2) XP, Rabbit mAb), Blue – Cell nuclei (DAPI)

Tight Junctions (Claudin-5)

Perfusion & Shear Stress

Experimental Examples:

By hosting the Fluidic Unit in the incubator, it is still possible to run perfusion assays directly on the microscope. A defined temperature and CO2 concentration is assured, as the fluidic reservoirs are inside the incubator at all times.

Claudin-5, a tight junction protein, can be found at the cell-cell contact zone when the cells are flow-conditioned for five days. This shows that the impact of the mechanical shear stress is crucial for the differentiation of the cell layer.

HUVEC, flow-conditioned 10 dyn/cm², 5 days µ-Slide I 0.4 Luer, ibiTreat

HUVEC, static culture 0 dyn/cm², 5 days µ-Dish 35 mm, ibiTreat

Golgi Apparatus

Red – F-Actin (Phalloidin-Alexa 633), Green – VE-Cadherin (VE-Cadherin (D87F2) XP), Blue – Cell nuclei (DAPI)

Von-Willebrand-Factor Left: The Golgi apparatus is localized along the direction of flow. Red – Golgi apparatus (Anti-Human-Golgin97, mAb, CDF4), Green – F-Actin (PhalloidinAlexa 488), Blue – Cell nuclei (DAPI)

HUVEC, flow-conditioned 10 dyn/cm², 4 days µ-Slide I 0.4 Luer, ibiTreat

Right: The von-Willebrand-Factor (vWF) is a typical endothelial cell membrane marker. When the cells are exposed to flow, the vWFmultimers elongate to rods that are sticking to the cell membrane. Green – von-Willebrand-Factor (AntiHuman-von-Willebrand-Factor IgG), Blue – Cell nuclei (DAPI)

HUVEC, flow-conditioned 10 dyn/cm², 5 days µ-Slide VI 0.4, ibiTreat

123


ibidi Pump System

NOTE: Custom specific slides can also be used when utilizing the Luer adapters.

Suitable µ-Slides The following ibidi µ-Slides can be attached to the ibidi Pump System: Low shear stress

Medium shear stress

High shear stress

• µ-Slide I Luer • µ-Slide I 0.8 Luer

• • • •

• • • •

Functional Cell-Based Assays

0.6

µ-Slide I Luer µ-Slide III 0.4 µ-Slide VI 0.4 µ-Slide y-shaped 0.4

µ-Slide I 0.1 Luer µ-Slide I 0.2 Luer µ-Slide III 0.1 µ-Slide VI 0.1 Perfusion Set

Perfusion Sets and µ-Slide Selection Overview:

Blue (ID 0.8 mm)

Low shear stress

Red (ID 1.6 mm)

White Yellow / Green Yellow (ID 0.8 mm) (ID 1.6 mm) (ID 0.5 mm)

X

X X

High shear stress Long tubes for microscopy (50 cm) Working volume (ml) Standard reservoir* (ml)

Black (ID 0.5 mm)

X X

X

X

X X

11.3

12.3

11.7

13.6

2.5

2.7

10

10

10

10

2

2

* By changing the reservoir, different working volumes can be achieved. Reservoirs are available with a syringe volume of 2, 12, and 50 ml.

Details: Perfusion Set blue (#10961) Inner diameter: 0.8 mm Total working volume: 11.3 ml

Tube length: 15 cm Dead volume of tubing: 0.5 ml Flow rate [ml / min]

Shear stress [dyn / cm²]

Growth area [cm²]

Channel volume [µl]

MIN

MAX

MIN

MAX

µ-Slide I 0.2 Luer

2.5

50

0.83

11.53

4.26

59.14

µ-Slide I

Luer

2.5

100

1.12

15.30

1.47

20.14

µ-Slide I 0.6 Luer

2.5

150

1.13

15.58

0.68

9.36

µ-Slide I

2.5

200

1.15

15.91

0.40

5.53

0.4

0.8

µ-Slide VI

Luer

0.4

µ-Slide y-shaped Without slide

0.6

30

1.12

15.25

1.97

26.86

2.8

110

1.08

14.50

2.46

32.98

-

(75)

1.18

16.51

-

-

Perfusion Set red (#10962) Inner diameter: 1.6 mm Total working volume: 12.3 ml

Flow rate [ml / min]

Shear stress [dyn / cm²]

Growth area [cm²]

Channel volume [µl]

MIN

MAX

MIN

MAX

µ-Slide I 0.2 Luer

2.5

50

1.28

19.11

6.57

98.02

µ-Slide I 0.4 Luer

2.5

100

2.65

33.17

3.49

43.66

µ-Slide I 0.6 Luer

2.5

150

2.74

34.98

1.65

21.02

2.5

200

2.75

35.14

0.96

12.21

µ-Slide VI 0.4

0.6

30

2.76

35.33

4.86

62.24

µ-Slide y-shaped

2.8

110

2.59

32.12

5.89

73.05

-

(75)

2.79

35.86

-

-

µ-Slide I

124

Tube length: 15 cm Dead volume of tubing: 1.5 ml

0.8

Luer

Without slide


Perfusion Set white (#10963) Inner diameter: 0.8 mm Total working volume: 11.7 ml

Tube length: 50 cm Dead volume of tubing: 0.9 ml Flow rate [ml / min]

Shear stress [dyn / cm²]

Channel volume [µl]

MIN

MAX

MIN

MAX

Luer

2.5

µ-Slide I 0.4 Luer

2.5 2.5 2.5 0.6 2.8 -

50 100 150 200 30 110 (75)

0.42 0.57 0.61 0.64 0.63 0.58 0.61

7.15 8.75 9.40 10.11 9.77 8.94 9.43

2.15 0.75 0.37 0.22 1.11 1.32 -

36.67 11.52 5.65 3.51 17.21 20.33 -

µ-Slide I

0.2

µ-Slide I 0.6 Luer µ-Slide I 0.8 Luer µ-Slide VI 0.4 µ-Slide y-shaped Without slide

Perfusion Set yellow / green (#10964) Inner diameter: 1.6 mm Total working volume: 13.6 ml

MIN

MAX

MIN

MAX

50 100 150 200 30 110 (75)

1.03 1.91 1.98 2.13 2.06 1.94 2.44

17.00 25.95 27.44 30.25 28.95 26.60 36.02

5.28 2.51 1.19 0.47 3.63 4.41 -

87.20 34.16 16.49 10.51 51.00 60.50 -

Luer

2.5

µ-Slide I

0.4

Luer

2.5 2.5 2.5 0.6 2.8 -

µ-Slide VI 0.4 µ-Slide y-shaped Without slide

Shear stress [dyn / cm²]

Channel volume [µl]

µ-Slide I

µ-Slide I 0.8 Luer

Flow rate [ml / min]

Growth area [cm²] 0.2

µ-Slide I 0.6 Luer

Tube length: 50 cm Dead volume of tubing: 2.8 ml

Perfusion & Shear Stress

Growth area [cm²]

Perfusion Set yellow (#10965)* Inner diameter: 0.5 mm Total working volume: 2.5 ml

µ-Slide I 0.1 Luer µ-Slide III 0.1 µ-Slide VI 0.1

Tube length: 15 cm Dead volume of tubing: 0.5 ml Flow rate [ml / min]

Shear stress [dyn / cm²]

Growth area [cm²]

Channel volume [µl]

MIN

MAX

MIN

MAX

2.5 0.43 0.17

25 4.5 1.7

0.07 0.06 0.08

1.35 1.08 1.44

1.42 6.40 8.54

27.34 115.27 153.69

Perfusion Set black (#10966)* Inner diameter: 0.5 mm Total working volume: 2.7 ml

µ-Slide I 0.1 Luer

µ-Slide III 0.1 µ-Slide VI 0.1

Tube length: 50 cm Dead volume of tubing: 0.7 ml Flow rate [ml / min]

Shear stress [dyn / cm²]

Growth area [cm²]

Channel volume [µl]

MIN

MAX

MIN

MAX

2.5 0.43 0.17

25 4.5 1.7

0.03 0.03 0.03

0.61 0.52 0.62

0.61 3.20 3.20

12.36 55.50 66.17

* Perfusion Set only works with PumpControl version 1.5.0 or higher and ibidi Pump with hardware version 1.02 or higher and firmware version 1.10 or higher.

125


KD Scientific Syringe Pumps Legato 100

Syringe pumps for flow assays, offered by ibidi in cooperation with KD Scientific

Functional Cell-Based Assays

• A broad range of syringe pumps for varied applications and budgets • Accurate delivery of fluids for the laboratory environment • Easy to use through a touch screen user interface Legato 270

Applications • Rolling and adhesion of suspended cells on substrates • Stop flow experiments • Drug infusion KD Scientific syringe pumps provide an accurate delivery of fluids for the laboratory environment. The series is divided into an economical line of unidirectional infusion pumps, and a high-end line of syringe pumps that have the capability to infuse and withdraw. Minimum and maximum flow rates depend on the size of the syringe being used. Infusion Pumps KD Scientific infusion pumps are ideal for the delivery of accurate and precise amounts of fluids to a multitude of applications. These include the injection of calibrant into a mass spectrometer or reaction chamber, the long-term drug delivery to animals, and general infusion applications. Infusion / Withdrawal Pumps Infuse and withdraw capabilities provide maximum flexibility to a variety of applications, such as the automatic withdrawal of samples and the unattended filling of syringes at very low flow rates. Push / Pull Syringe Pumps These proven KDS pumps provide simultaneous infusion and withdrawal with opposing syringes on a single drive. Each one has been modified to hold an additional syringe, allowing one syringe to infuse, while the second one simultaneously withdraws. Software The ADAGIO Software is for Legato Pumps only. Technical Details: For detailed information on the individual syringe types, please contact us directly. Ordering Information:

126

Cat. No.

Description

10940

Syringe Pump KDS 100, Infuse only, 1 syringe

Pcs. / Box 1

Cat. No. Description 11956

Syringe Pump Legato 200, Infuse only, 2 syringes

Pcs. / Box

11970

Syringe Pump Legato 100, Infuse only, 1 syringe

1

11957

Syringe Pump Legato 210, Infuse / Withdraw, 2 syringes 1

11958

Syringe Pump Legato 210 P, Infuse / Withdraw, programmable, 2 syringes

1 1

11971

Syringe Pump Legato 101, Infuse only, 2 syringes

1

11968

Syringe Pump Legato 110, Infuse / Withdraw, programmable, 1 syringe

1 11959

Syringe Pump Legato 270, Push / Pull, 4 syringes

1

11967

Syringe Pump Legato 111, Infuse / Withdraw, programmable, 2 syringes

1

11960

Syringe Pump Legato 270 P, Push / Pull, programmable, 4 syringes

1

11972

Syringe Pump Legato 130, Infuse/Withdraw, 1 syringe

1

11980

ADAGIO Software for Legato Pumps

1

11969

Syringe Pump Legato 180, Infuse / Withdraw, programmable, 2 syringes

1


µ-Galaxy Incubator Incubator for µ-Slides

• A small sized system with a capacity of only 14 liters • Robust in routine applications

• Independent flow assays using the ibidi Pump System (see page 118) • ECIS measurements (see page 138) • Separated assays

Technical Features • Input pressure of only 0.35 bar – saves gases • Full CO2 and heat control • Sealable outlet for cables and tubes • Relative humidity up to 95 % Cultivation of cells normally occurs at 37° C and 5 % CO2 in cell culture incubators. However, during ECIS measurements, or when working with ibidi µ-Slides, it is necessary to manipulate cells frequently. As each opening affects the climate inside the incubator, and in order to prevent any contamination of the regular cell culture, we recommend running these experiments separately in a dedicated µ-Galaxy incubator.

Perfusion & Shear Stress

Applications

The µ-Galaxy incubator is characterized by its small size and a capacity of only 14 liters. It is a compact system that occupies minimal lab space and requires only small amounts of CO2. The rear of the µ-Galaxy incubator includes a sealable outlet for easy installation of exterior cables or tubes. The incubator contains two trays for racks, µ-Slides, or ECIS slide holders.

Example Setup for perfusion experiments using two Fluidic Units (see page 119)

Ordering Information:

Specifications / Content:

Cat. No.

Description

Pcs. / Box

10991

µ-Galaxy cell culture incubator, small capacity system (14 l), specially suited for setups employing up to 2 Fluidic Units, or for separated setups like ECIS measurements

1

Volume

14 l

Outer dimensions 31 x 33 x 43 cm width x depth x height Inner dimensions 23 x 21 x 30 cm width x depth x height Outlet Ø

25 mm

Input pressure

0.35 bar (5 psi)

127


µ-Slide I Luer Family Channel slides with different heights, volumes, and coatings specially suited for flow applications

• Large area of uniform shear stress

Functional Cell-Based Assays

• Easy connection using Luer adapters • Homogeneous cell distribution with defined optical pathway

Applications • Adherent cells under flow conditions • Cell culture (static or stop-flow) • 3D cell culture in gels brought into the channels • High resolution microscopy of living and fixed cells

200 µm

Technical Features • Standard format with thin bottom for low or high magnification microscopy (up to 100 x)

Flow cultivation

• Large observation area for microscopy • Channel volumes of 25 µl, 50 μl, 100 μl, 150 μl, or 200 μl • Defined shear stress and shear rate levels • Easy connection to tubes and pumps using Luer adapters

200 µm

• Available as a variety pack containing all heights • Fully compatible with the ibidi Pump System (see page 118)

Static cultivation

Cross Section of the Channel: Same Slide – Different Channel Height and Volume µ-Slide I 0.1 Luer *

µ-Slide I 0.2 Luer*

100 µm / 25 µl

200 µm / 50 µl

µ-Slide I 0.4 Luer

µ-Slide I 0.6 Luer

400 µm / 100 µl

µ-Slide I 0.8 Luer

600 µm / 150 µl

800 µm / 200 µl

* not recommended for static culture over more than 6 hours

Ordering Information: µ-Slide I

0.1 Luer

Coating

Cat. No.

0.4 Luer

0.6 Luer

0.8 Luer

80166

80176

80186

80196

15

Collagen IV, sterile

81122

80162

80172

80182

80192

15

Fibronectin, sterile*

81123

80163

80173

80183

80193

15

Poly-L-Lysine, sterile

80164

80174

80184

80194

15

Poly-D-Lysine, sterile*

80165

80175

80185

80195

15

81121

80161

80171

80181

80191

15

ibiTreat, tissue culture treated, sterile

hydrophobic, uncoated, sterile * available only on request

128

Specifications:

0.2 Luer

Pcs. / Box

Channel length

50 mm

Channel width

5 mm

Adapters

Female Luer

Volume per reservoir

60 µl

Growth area per channel

2.5 cm2

Coating area: 5.1 / 5.2 / 5.4 / 5.6 / 5.8 cm2 Bottom: ibidi Standard Bottom

Cat. No.

Description

81106

µ-Slide I Luer variety pack, ibiTreat, tissue culture treated, sterile, 4 times each channel height, 200 / 400 / 600 / 800 µm

Pcs. / Box 16

81101

µ-Slide I Luer variety pack, hydrophobic, uncoated, sterile, 4 times each channel height, 200 / 400 / 600 / 800 µm

16


sticky-Slide I Luer A bottomless channel slide used for perfusion applications with a self-adhesive underside

• Versatile – i.e., permits the mounting of a variety of bottom materials • Available in various channel heights from 0.1 to 0.8 mm

• Inserting materials or tissue into perfusion channels • Running of shear stress experiments on any substrate • Usable with a range of bottom materials, such as plastic sheets, silicon chips, and glass slides

Technical Features • Cell culture tested biocompatible adhesive • Adheres to all flat surfaces, even wet surfaces • Includes female Luer adapters for connection to tubing and pump systems • Suitable coverslips available • Specifications identical to those of the µ-Slide l Luer (see page 128), except that it has no bottom Example 1: Sample Insertion into a Channel for Perfusion Studies

Example 2: Setup of a Sample Squeezed into a Channel

Perfusion & Shear Stress

Applications

sticky-Slide with channel

Prepare your sample

Sample

Sample preparation

Coverslip

Remove the protection film

Pressing and sample fixation Position your sample Assemble all parts with a coverslip

Ordering Information: Cat. No. Description 81128

Specifications: Pcs. / Box

sticky-Slide I 0.1 Luer, sterile 15

80168 sticky-Slide I 0.2 Luer, sterile 15 80178

sticky-Slide I 0.4 Luer, sterile 15

80188 sticky-Slide I 0.6 Luer, sterile 15 80198 sticky-Slide I 0.8 Luer, sterile 15 10812

Assembled channel

Coverslips for sticky-Slides, 100 No. 1.5H (170 µm +/- 5 µm) D 263 M Schott glass, unsterile, 25 mm x 75 mm

sticky-Slide I

0.1 Luer

0.2 Luer

0.4 Luer

0.6 Luer

0.8 Luer

Volume of the channel

25 µl

50 µl

100 µl

150 µl

200 µl

Height of the channel

100 µm

200 µm

400 µm

600 µm

800 µm

Channel length Adapters Volume per reservoir Growth area per channel Bottom

50 mm Female Luer 60 µl 2.5 cm2 none

129


µ-Slide VI 0.1 | µ-Slide III 0.1 A three or six channel µ-Slide for parallel flow assays and immunofluorescence that uses a minimal amount of reagents and medium

Functional Cell-Based Assays

• Perfect for rolling and adhesion assays on cell monolayers or protein coatings • Minimal amounts of cells and / or liquids needed • Medium-to-high shear stress that is applicable with small volumes

Applications • Flow assays that use a minimum of cells and / or liquids (mouse model) ibidi APPLICATION NOTE

• Parallel shear stress applications • Immunofluorescence microscopy after perfusion • High-resolution microscopy of cells under shear stress

Find detailed information on shear stress / shear rates and flow rates in our Application Note

Technical Features

Shear Stress and Shear Rates (AN 11).

• Three or six parallel channels on one slide • Small sample amounts • Not for use in static culture • Easy connection to tubes and pumps using a female Luer adapter • Defined shear stress and shear rate levels

Adhesion Assay

• µ-Slide VI 0.1: Smallest ibidi channel slide with highest shear stress • µ-Slide III 0.1: 1 mm scale for sample relocation

Leukocytes

Cell / blood collection

Cell processing

Cell culture

Endothelial Cells

Cell preparation, e.g., from umbilical cord

Perfusion experiment

Cell culture

Specifications:

130

Cell seeding

µ-Slide VI 01

µ-Slide III 01

Ordering Information:

Adapters

Female Luer

Female Luer

Coating

Volume per reservoir

60 µl

60 µl

Number of channels

6

3

ibiTreat, tissue culture treated, sterile

Volume of each channel

1.7 µl

4.5 µl

Collagen IV, sterile

µ-Slide VI 01 µ-Slide III 01 Cat. No. 80666

Pcs. / Box –

15

80662

80332

15

80663

80333

15

Height of channels

0.1 mm

0.1 mm

Fibronectin, sterile*

Length of channels

17 mm

45 mm

Poly-L-Lysine, sterile

80664

15

Width of channels

1 mm

1 mm

Poly-D-Lysine, sterile*

80665

15

Growth area per channel

0.17 cm2

0.43 cm²

hydrophobic, uncoated, sterile

80661

80331

15

Coating area per channel

0.34 cm2

0.86 cm²

Distance of scale bars

1 mm

Bottom:

ibidi Standard Bottom

* available only on request


µ-Slide VI 0.4 | µ-Slide III 0.4 (coming soon)

N

EW

A three or six channel µ-Slide suitable for flow experiments and for immunofluorescence assays

• Optimal phase contrast under both static and flow conditions • Homogeneous cell distribution over the channel surface, regardless of handling practices

Perfusion & Shear Stress

• Cost-effective experiments with small numbers of cells and low volumes of reagents

Applications • Real-time imaging under either static or flow conditions • Parallel screenings using multi-channel pipettes • Immunofluorescence assays and live cell imaging

Technical Features • 30 µl channel volume, which saves on reagent consumption • Easy connection to existing tubes and pumps via female Luer adapter • Fully compatible with high resolution fluorescence microscopy • Defined shear stress and shear rate levels

NOTE

ibidi TIP

Choosing the Right Channel for Flow Applications

Static Cultures versus Flow Applications

• For flow assays employing the smallest volumes and high values of shear stress: use a 0.1 or 0.2 mm channel

• Low walled channels are more suitable for flow applications.

• For wide ranging shear stress values: use a 0.4 mm channel

• High walled channels are more suitable for static cell culture.

• For controlling low values of shear stress (< 2 dyn / cm²): use a 0.6 or a 0.8 mm channel

Specifications:

µ-Slide VI 04

µ-Slide III 04

Ordering Information:

µ-Slide VI 04 µ-Slide III 04

Adapters

Female Luer

Female Luer

Coating

Volume per reservoir

60 µl

60 µl

15

6

3

ibiTreat, tissue culture treated, sterile

80606

Number of channels Volume of each channel

30 µl

Coming soon

Collagen IV, sterile

80602

15

Height of channels

0.4 mm

0.4 mm

80342 coming soon

Length of channels

17 mm

50 mm

Fibronectin, sterile*

80603

80343 coming soon

15

Cat. No.

Pcs. / Box

Width of channels

3.8 mm

Coming soon

Poly-L-Lysine, sterile

80604

15

Growth area per channel

0.6 cm2

Coming soon

Poly-D-Lysine, sterile*

80605

15

Coating area per channel

1.20 cm2

Coming soon

hydrophobic, uncoated, sterile

80601

80341 coming soon

15

Bottom:

ibidi Standard Bottom * available only on request

131


µ-Slide y-shaped A flow through μ-Slide for bifurcation studies and blood vessel simulation

• Perfect simulation of live conditions in branching blood vessels

Functional Cell-Based Assays

• Allows flows in two different directions under different angles

Applications • Rolling and adhesion of leukocytes on endothelial cells cultured under flow ibidi APPLICATION NOTE

• Simulation of the bifurcation of blood vessels for arteriosclerosis research • Cell-cell interaction studies and cell-drug interaction screenings under flow conditions

Find more information in AN 18: Shear Stress and Shear Rates in µ-Slide y-shaped

Technical Features • 30° and 45° angles to simulate the bifurcation of blood vessels • Easy connection to tubes and pumps • Fully compatible with high resolution microscopy and immunofluorescence staining • Defined shear stress and shear rate levels • The flow is always laminar, i.e. turbulent flows* are not possible * For simulation of turbulences we recommend oscillating the flow using the ibidi Pump System (see page 118).

Non-Uniform, Laminar Shear Stress

Two Flow Directions Possible

45°

30°

Specifications: Adapters

132

Ordering Information: Female Luer

Cat. No.

Description

80126

µ-Slide y-shaped, ibiTreat, tissue culture treated, sterile

Pcs. / Box 15

80122

µ-Slide y-shaped, Collagen IV, sterile

15

80123

µ-Slide y-shaped, Fibronectin, sterile*

15

Volume per reservoir

60 µl

Volume of the channel

110 µl

Height of the channel

0.4 mm

Growth area

2.8 cm2

80124

µ-Slide y-shaped, Poly-L-Lysine, sterile

15

Coating area

5.6 cm2

80125

µ-Slide y-shaped, Poly-D-Lysine, sterile*

15

80121

µ-Slide y-shaped, hydrophobic, uncoated, sterile

15

Bottom: ibidi Standard Bottom

* available only on request


Elbow Luer Connector Connects female Luers with 0.8 or 1.6 mm ID tubing • Saves maximum space on the microscope

Male Luer Adapter

General

Suitable for Female Luer adapters

Total height

18 mm

ID

Angle

90°

2 mm

Cat. No. Description

Temp. stability Up to 125 °C (autoclav.) Material

Polypropylene

Suitable for Flexible tubes, ID 0.8-1.6 mm

Color

White

ID

Max. pressure

500 mbar

0.8 mm

Luer Connector Male Connects female Luers with 0.5, 0.8 or 1.6 mm ID tubing • Straight connector used for vertical tube mounting

Male Luer Adapter

General

Suitable for Female Luer adapters

Total length

18 mm

ID

Angle

Straight (0°)

3 mm

Cat. No. Description

Temp. stability Up to 125 °C (autoclav.) Single Barb

Material

Polypropylene

Suitable for Flexible tubes, ID 0.5-1.6 mm

Color

Natural

ID

Max. pressure

500 mbar

1 mm

10824

Pcs. / Box

Luer Connector Male, natural Polypropylene, sterile

25

Luer Lock Connector Male

Perfusion & Shear Stress

Single Barb

Pcs. / Box

10802 Elbow Luer connector, 50 male Luer to single barb, elbow style for all µ-Slides Luer, white Polypropylene, sterile

Connects female Luers with 0.5, 0.8 or 1.6 mm ID tubing • Provides an easy closing and re-opening mechanism

Male Luer Lock

General

Suitable for Female Luer or Luer Lock adapters

Total length

15 mm

Angle

Straight (0°)

ID

3 mm

Cat. No. Description

Temp. stability Up to 125 °C (autoclav.)

Single Barb

Material

Polypropylene

Suitable for Flexible tubes, ID 0.5-1.6 mm

Color

Natural

ID

Max. pressure

500 mbar

1 mm

Pcs. / Box

10826 Luer Lock Connector Male, natural Polypropylene, sterile

25

Luer Lock Connector Female Connects male Luers with 0.5, 0.8 or 1.6 mm ID tubing • Provides an easy closing and re-opening mechanism

Female Luer Lock

General

Suitable for Male Luer or Luer Lock adapters

Total length

16 mm

Angle

Straight (0°)

ID

4 mm

Cat. No. Description

Temp. stability Up to 125 °C (autoclav.)

Single Barb

Material

Polypropylene

Suitable for Flexible tubes, ID 0.5-1.6 mm

Color

Natural

ID

Max. pressure

500 mbar

1 mm

Pcs. / Box

10825 Luer Lock Connector Female, natural Polypropylene, sterile

25

133


Female Luer Lock Coupler Connects two male Luer Lock adapters • Coupler for a variety of fluidic setups Cat. No. Description

Pcs. / Box

Functional Cell-Based Assays

10823 Female Luer Lock Coupler, 25 natural Polypropylene, sterile

Female Luer Lock Coupler Suitable for

Male Luer or Luer Lock adapters

General Total length ID Temp. stability Material Color Max. pressure

16 mm 4 mm Up to 125 °C (autoclav.) Polypropylene Natural 500 mbar

Luer Plug Male Closes female Luer adapters, such as those found on an ibidi µ-Slide • Simply and tightly seals ibidi channel µ-Slides with Luer adapters Cat. No. Description

Pcs. / Box

10822 Luer Plug Male, natural Polypropylene, sterile

25

Luer Plug Male Suitable for

Female Luer adapters

General Total height Temp. stability Material Color Max. pressure

15 mm Up to 125 °C (autoclav.) Polypropylene Natural 500 mbar

In-line Luer Injection Port An in-line injection port including a silicone septum, used to inject liquids into a running flow experiment • Allows fluids to be injected without stopping the flow Cat. No. Description

Pcs. / Box

10820 In-line Luer injection port with silicone septum, single packed, sterile

25

Male Luer Lock Adapter Suitable for Female Luer Lock adapters 2.5 mm ID Female Luer Lock Adapter Suitable for Male Luer Lock adapters 4 mm ID

General Total length Temp. stability Material Color Max. pressure

38.5 mm Up to 90 °C (not autoclav.) Polycarbonate / Silicone Clear 500 mbar

Hose Clip A hose clip used to temporarily or permanently closing silicone tubes • Perfect for all flow applications Cat. No. Description 10821

Hose clip

Pcs. / Box 5

Hose Clip Suitable for

Silicone tubing from ID 0.5 mm to 1.6 mm

General Total length Sealing length Temp. stability Material Color

90 mm 70 mm Up to 80 °C (not autoclav.) Polyamide White

Tube Adapter Set Connects existing equipment with ibidi µ-Slides

N EW

• Autoclavable tubes and connectors

Cat. No. Description 10831 Tube Adapter Set, sterile

134

Pcs. / Box 6x2

Male Luer Adapter Suitable for Female Luer adapters 0.8 / 2 mm ID Single Barb Suitable for Flexible tubes, ID 3.2 mm ID 1.1 / 1.7 mm

General ID Tubing Total length Angle Temp. stability Material Color Max. pressure

1.6 mm 20 cm 90° Up to 125 °C (autoclav.) Polypropylene & Silicone Tubing Natural 500 mbar


Serial Connector Connects two µ-Slides with female Luers in a serial manner • Allows for the connection of fluids between two µ-Slides for serial experiments

Angle

General Suitable for

Straight (0°)

Temp. stability Up to 125 °C (autocl.) Material

Polypropylene & Silicone Tubing

ID Tubing

1.6 mm

Color

Natural

Total length

60 mm

Max. pressure

500 mbar

Cat. No. Description

Pcs. / Box

10830 Serial connector for µ-Slides, sterile

6 x 2

Y Tube Fitting 0.8 mm / 1.6 mm Y-style splitter for silicone tubing • Permits easy setup of complex fluidic experiments

Y Tube Fitting 0.8 mm

General

Suitable for Flexible tubes, ID 0.5-0.8 mm

Total length

11 mm / 16.5 mm

ID

Angle

60° between branches

1 mm

Cat. No. Description

Temp. stability Up to 125 °C (autoclav.) Y Tube Fitting 1.6 mm

Material

Polypropylene

Suitable for Flexible tubes, ID 1.6 mm

Color

Natural

ID

Max. pressure

500 mbar

1.8 mm

Pcs. / Box

10827 Y Tube Fitting 0.8 mm ID, natural Polypropylene, sterile

25

10828 Y Tube Fitting 1.6 mm ID, natural Polypropylene, sterile

25

Fitting Reducer 0.5 to 1.6

Perfusion & Shear Stress

Female Luer or Female Luer Lock adapters

For adapting 0.5 or 0.8 mm ID tubing to 1.6 mm ID silicone tubing • Provides simple tube-to-tube connections for sophisticated fluidic setups using different tube sizes

0.5 mm Barb

General

Suitable for Flexible tubes, ID 0.5 to 0.8 mm

Total length

15 mm

ID

Angle

Straight (0°)

1 mm

Cat. No. Description

Temp. stability Up to 125 °C (autoclav.) 1.6 mm Barb

Material

Polypropylene

Suitable for Flexible tubes, ID 1.6 mm

Color

Natural

ID

Max. pressure

500 mbar

1.8 mm

Pcs. / Box

10829 Fitting Reducer 0.5 to 1.6, 25 natural Polypropylene, sterile

Silicone Tubing Biocompatible silicone tubing used for a range of flow applications • Available in a variety of inner diameters for use in various flow experiments Tubing 0.5 mm

Tubing 0.8 mm

Tubing 1.6 mm

Cat. No. Description

Inner diameter

0.5 mm

0.8 mm

1.6 mm

1

Wall thickness

0.8 mm

1 mm

0.8 mm

10840 Silicone Tubing 0.5 mm ID, 5 m, silicone, sterile

Temp. stability

Up to 125 °C (autoclav.) Up to 125° C (autoclav.) Up to 125° C (autoclav.)

10841

1

Material

Silicone

Silicone

Silicone

Silicone Tubing 0.8 mm ID, 5 m, silicone, sterile

Color

Natural

Natural

Natural

1

Max. pressure

500 mbar

500 mbar

500 mbar

10842 Silicone Tubing 1.6 mm ID, 5 m, silicone, sterile

Pcs. / Box

135


Functional Cell-Based Assays

Customer Feedback

„I work with the ibidi slides because they are superior to any other product of this kind on the market. They are easy to use, give consistent results, economical and are suitable for a wide range of applications. The flow slides have without a doubt transformed my labs research and made many new experiments possible. Well done ibidi! I am indeed extremely happy with the ibidi products.” Dr. Thomas A.J. McKinnon BSc, PhD Department of Hematology Imperial College London UK

“We’ve been working with the ibidi Pump System and Labware for over 5 years now and have recommended it to numerous colleagues as well. The plastics are ideal for cell culture and all forms of imaging, including automated imaging (easy to focus) and all sorts of fluorescence (clear, optically superb plastics). The ibidi Pump System in fact made the endothelial cell under flow the default of our lab! And above all, the technical support is always fast and efficient.” Dr. Nynke van den Akker Department of Cardiology Maastricht University Maastricht The Netherlands

“The µ-Slide I Luer facilitates the culture of endothelial cells under defined complex flow patterns. We are particularly interested in the effects of oscillatory shear stress on redox signaling pathways, and the ibidi Air Pump and Fluidic Units are ideal systems for this purpose. The µ-Slides are convenient for both live and fixed cell imaging as well as the collection of protein and RNA samples and other cell analyses.”

Shane R. McSweeney Cardiovascular Division, BHF Centre of Research Excellence, School of Medicine King’s College London United Kingdom

References

136

Recombinant Lectin-Like Domain of Thrombomodulin Suppresses Vascular Inflammation by Reducing Leukocyte Recruitment via Interacting with Lewis Y on Endothelial Cells W.-L. Lin, C.-F. Chang, C.-S. Shi, G.-Y. Shi and H.-L. Wu Arteriosclerosis, Thrombosis, and Vascular Biology, 2013, 10.1161/ atvbaha.113.301221

Transglutaminase activity regulates atherosclerotic plaque composition at locations exposed to oscillatory shear stress H. L. Matlung, A. E. Neele, H. C. Groen, K. van Gaalen, B. G. Tuna, A. van Weert, J. de Vos, J. J. Wentzel, M. Hoogenboezem and J. D. van Buul Atherosclerosis, 2012, 10.1016/j. atherosclerosis.2012.07.044

Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics E. Liaskou, H. W. Zimmermann, K. K. Li, Y. Htun Oo, S. Suresh, Z. Stamataki, O. Qureshi, P. F. Lalor, J. Shaw and W. Syn HEPATOLOGY, 2013, 10.1002/ hep.26016

Stabilization of Brain Microvascular Endothelial Barrier Function by Shear Stress Involves VE cadherin Signaling Leading to Modulation of pTyr Occludin Levels T. G. Walsh, R. P. Murphy, P. Fitzpatrick, K. D. Rochfort, A. F. Guinan, A. Murphy and P. M. Cummins Journal of Cellular Physiology, 2011, 10.1002/jcp.22655

Primary Cilia Regulate mTORC1 Activity and Cell Size Through Lkb1 C. Boehlke, F. Kotsis, V. Patel, S. Braeg, H. Voelker, S. Bredt, T. Beyer, H. Janusch, C. Hamann, M. Gödel, K. Müller, M. Herbst, M. Hornung, M. Doerken, M. Köttgen, R. Nitschke, P. Igarashi, G. Walz and E. W. Kuehn Nature Cell Biology, 2010, 10.1038/ncb2117


Electric Cell Substrate Impedance Sensing (ECIS™) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 ECIS Instrument Selection Guide . . . . . . . . . 143 ECIS Cultureware 8 well . . . . . . . . . . . . . . . . 144 ECIS Cultureware 96 well . . . . . . . . . . . . . . . 146 ECIS Transwell Measurement System . . . . . 146

Impedance-Based Cell Assays

IMPEDANCE-BASED CELL ASSAYS . . . . . 138

137


Impedance-Based Cell Assays

Impedance-Based Cell Assays

6000

12,00

6000 5000

10,00

4000 3000 2000 1000

5000

0

0

5

10

kOhm

7000

15

Time (hrs)

4000 3000

C

2000 1000 0 0

5

10

15

Time (hrs)

8,00 6,00 4,00

7

2,00

6 5 4

0,00

3

1

2 1

2

5

10

15

Time (hrs)

ECIS Array (page 144)

Experimental Results from ECIS System (page 142)

Membrane capacitance

Barrier function

R = Resistance C = Capacitance f = Frequency of AC current

5

6

Data Analysis

Electric Cell Substrate Impedance Sensing (ECIS™; see page 142) measures the change in impedance of a small electrode to AC current flow over time. The current flows between a 250 µm diameter electrode and a larger counter electrode, using standard culture medium as the electrolyte.

α

When using the ECIS Z Theta System (see page 143), impedance can be divided into two sets of data – one due to pure resistance (R) and the other due to capacitance (C). Capacitance measured at high frequencies (> 40 kHz) can be used to ascertain what fraction of the substrate is covered by cells.

Cm

Basic Principle of ECIS Measurement

4

Rb 3

2

1

1

2

3

4

5

Time [h]

Cell seeding

α Cm Membrane Capacitance Rb Resistance between cells α Cleft impedance parameter

Impedance (ohm)

Rb

Cm

138

5

After inoculating cells into a well, cells anchor and spread on the base of the well and onto the active 250 µm electrode. The insulating plasma membranes of the cells on the electrode constrain the electrode’s electrical current and force the current to flow in regions beneath and between the cells. This convoluted current path causes large changes in the measured impedance. With the confluent cell layer in place, the resistance reaches a plateau. The AC current used to make the impedance measurements has no detectable effects upon the cells. This means that the measurements are non-invasive.

Impedance (Z):

0

4

Working Principle Electrode

0

3

µM

0 0

Cell layer

Norm. Parameter

Resistance (Ohm)

Impedance (Ohm)

PC

7000

Capacitance (nF)

Experimental Workflow

R

Uncovered electrode surface

z=z0

Cell manipulation Covered electrode surface

z=z1

Time [h]

After cell manipulation

z=z2


Wounding and Cell Migration Impedance Measurements The ECIS Wound Healing Assay replaces the traditional “scratch” assay. Instead of disrupting the cell layer mechanically with a pipette tip and then following the migration of cells with a microscope, the ECIS system employs electric signals to both wound and monitor the healing process.

Electrical wounding is directed to a small population of cells in contact with the 250 µm electrode thus creating a well defined wound.

6000 5000 4000

10 %

3000

1%

0%

2000 1000 0

0

5

10

15

20

Time [h]

Wounding

Optical Measurements Optical measurements can easily be taken with ibidi’s Culture-Insert (page 94), which provides two culture reservoirs each separated by a 500 µm wall. Culturing cells in both reservoirs and then removing the insert, results in two well-defined cell patches. The assayspecific WimScratch Image Analysis (see page 156) can then be used to generate quantitative data based on image data acquired using video microscopy. 6

Weak Adhesion

5

C40 kHz [nF]

Cell Attachment Impedance Measurements The interaction between surfaces and cells is a main component of cell behavior in vivo and in vitro and therefore an area of major interest in cell biology studies. ECIS allows researchers to study and quantify the interaction of cultured cells with extracellular matrix proteins, and other macromolecules by taking continuous, real-time impedance measurements.

4 3

BSA

VN

2

LAM

1 0

Strong Adhesion

FN 0

5

10

15

20

Impedance-Based Chemotaxis Cell Assays

After wounding, the healthy cells around the electrode immediately migrate and replace the dead cells on the electrode. Depending on the fetal calf serum (FCS) concentration, the original plateau is once again reached after 10, 13 or 20 hours.

Controls Impedance [Ohm]

Experimental Example: Healing Function of Serum

Time [h]

MDCK II cells inoculated on electrodes pre-coated with various proteins, i.e.: FN Fibronectin, LAM Laminin, VN Vitronectin, BSA Bovine serum albumin

Optical Measurements Optical measurements of cell attachment rates are taken using ibidi’s µ-Slide VI 0.4 (see page 78) and µ-Slide Angiogenesis (see page 100). µ-Slide VI 0.4

µ-Slide Angiogenesis

Cell attachment after a predefined period of time (e.g., 4 hours) using different coatings and surface treatments.

Collagen IV Fibronectin Laminin at Tre ibi I en llag Co IV en llag Co tin ec ron Fib nin mi e La sin -Ly ly-L Po

Concentration

139


Experimental Example: Effect of Barrier Disruption on Endothelial Cells After Addition of Cytochalasin D

Impedance Measurements

2.0

1.5 400 1.0 200 0.5

0

25

50

75

C (40 kHz) [µF]

Z (400 Hz) [Ω]

600

100

Time [min]

Impedance-Based Cell Assays

Measurement and Modeling of Endothelial Cell Barrier Function

The capacitance (C) of the cell layer covering the electrode remains constant after disruption of the cell-cell contacts while impedance (Z) breaks down dramatically.

Measuring barrier function (i.e., the resistance of the paracellular pathway between cells) has proven to be a significant and rapidly growing application in ECIS technology. The use of ECIS technology to monitor barrier function was first demonstrated in 1992 in an experiment using bovine pulmonary endothelial cells exposed to thrombin. Recent data has suggested that various cellular effects can be monitored at different frequencies used during experimentation. While cell spreading can be analyzed at high frequencies, the formation or disruption of a barrier function is best described at low frequencies. For more information on this subject, please refer to: „Impedance Analysis of Cell Junctions” (Wegener, J.) In: Nanotechnology Ed. H. Fuchs. VCH Weinheim, 2009.

+CD Optical Measurements Optical measurements of barrier function are possible with all ibidi-µ-Slides and µ-Dishes when used in conjunction with fluorescent dyes.

Staining of ZO-1 protein in endothelial cells showing barrier disruption after addition of Cytochalasin D (CD).

Flow Assays Impedance Measurements in Combination with the ibidi Pump System Endothelial cells in vivo and in vitro respond to shear stress by activating intracellular signaling pathways, with morphological changes and gene expression. The shown experimental setup combines the ECIS system with the Pump System (page 118) to more closely simulate the in vivo environment.

Norm. Resistance

101

No Flow

Flow

HUVECs (Human umbilical vein endothelial cells) cultivated under flow conditions for 340 hours. Resistance was measured using the ECIS system in conjunction with an ECIS Flow Array (see page 146).

100 0

50

100

150

No flow

200

250

300

350

No flow

Flow (5 / 10 / 20 dyn / cm2)

Flow (20 dyn / cm2)

Time [h]

Optical Measurements Experimental Example: Morphological Changes in Cell Cytoskeleton after Cultivation under Flow Conditions Live cell imaging of HUVECs cultured under flow conditions (20 dyn / cm²) in µ-Slide I 0.4 Luer (see page 128) for 9 days. The cells were transduced with the adenoviral vector rAV CMV LifeAct-TagRFP (see page 33) 24 hours prior to running the experiment.

140


Impedance Measurements Studies measuring well-defined dose- and time-related toxic effects of chemicals often employ small numbers of cells during testing, and ECIS has been successfully used in such studies. ECIS effectively describes the relationship between dose and its effects on the exposed organism, e.g., comparisons of LD50 (Lethal Dose, 50 %) values, toxicological effects from heavy metals, and the effects of viral infections on cell populations.

4000

Resistance [ohm] 4 kHz

Toxicological Screening

3000

2000

1000

0 0

Control Undiluted 1 : 10

50

100

1 : 1 000 1 : 100 000 1 : 1 000 000

150

200

250

300

350

Time [h]

Cells challenged with decreasing concentrations of a virus

Optical measurements of toxicology are possible with all ibidi-µ-Slides and µ-Dishes.

Measurement of Metastatic Potential

14000

Control

Impedance Measurements Given that the ECIS system easily detects alterations in the endothelial cell layer, these changes can be monitored to look for correlations between changes in impedance and the metastatic potential of cancer cells. Experimental Example: The Dunning Prostatic Adenocarcinoma Series Metastatic cell lines were added to a confluent HUVEC (Human Umbilical Vein Endothelial Cells) cell layer, which resulted in a significant loss of resistance. This loss of resistance was due to the endothelial cell layer’s loss of integrity in response to the activities of the cancer cells. As a control, the same metastatic cells were heat killed (by keeping them 15 minutes at 56 °C) before adding them to another confluent HUVEC cell layer. No changes in resistance were detected in this instance, thus verifying that the assay was indeed perceiving biological activities.

Resistance [ohm]

12000 10000 8000

G 6000 4000

AT3 2000 2

4

6

8

10

12

14

16

Time [h]

Metastatic potential of two different cell lines: G and AT3

Impedance-Based Chemotaxis Cell Assays

Optical Measurements

Results: Cell Line G – weakly metastatic Cell Line AT3 – highly metastatic

Optical Measurements After adding metastatic cell lines to confluent cell layers, optical measurements under flow conditions are easily performed using any ibidi µ-Slide (e.g., µ-Slide I Luer, see page 128) in conjunction with the ibidi Pump System (see page 118). 20x

141


in cooperation with

Applied BioPhysics

www.biophysics.com

Electric Cell Substrate Impedance Sensing (ECIS™) A morphological biosensor used to measure cell behavior which employs a label-free, non-invasive method to electronically monitor cells grown in tissue culture

• Non-invasive cell-based assays in culture medium • Measurement of changes in cell morphology • Multiple, real-time measurements

Impedance-Based Cell Assays

8W1E

8W10E

8W10E+

Applications • Cell attachment and migration • Wound healing • Signal transduction • Cell behavior under flow • Measurement of metastatic potential • Measurement and modeling of endothelial cell barrier function • Toxicological screening The ECIS system represents a non-invasive approach to monitor living cells in vitro. It measures the change in impedance of a small electrode over time. The ECIS system gathers continuous real-time measurements without the need for any labels. Furthermore, since the weak AC currents used during measurement have no effect upon the cells, this method is totally non-invasive. There are two major systems: The ECIS Model Z and the ECIS Model Zθ (theta). The ECIS Model Z system monitors the impedance of small 250-micrometer diameter electrodes used as substrates for cell growth. The ECIS Model Zθ system interprets complex impedance as both resistance and capacitance, and can report these values as well as simple impedance. Both systems are available with either a 2 x 8 well or a 96 well array station that is ideal for high throughput applications. The array stations are hosted in an incubator so that cells are kept under ideal cultivation conditions at all times. For impedance measurements under flow conditions, combine the ECIS system with the Pump System to more closely simulate the in vivo environment.

ibidi TIP For Application Notes and more than 500 scientific publications using the ECIS technology please visit www.biophysics.com.

142


ECIS Instrument Selection Guide

Dimensions: 44 x 22 x 51 cm Weight: 18 kg

Cat. No. Description

Cat. No. Description

71616

71617

ECIS Model Z, which contains: ECIS Model Z station controller; Elevated Field Module (i.e., a pulse system for automated wound healing or electroporation studies); software for data gathering and analysis; laptop

ECIS Model Z Theta, which contains: ECIS Model Z Theta station controller; Elevated Field Module (i.e., a pulse system for automated wound healing or electroporation studies); software for data gathering and analysis, including model calculations; laptop

Measurement Specifications ECIS Z

Measurement Specifications ECIS Zθ

• Simple impedance Z • Well capacity: 16 or 96 wells • Maximum data acquisition rate (single frequency): 5 point / sec • Multi-frequency measurements: 11 frequencies ranging from 500 Hz to 64 kHz (10 seconds per well acquisition rate) • Wounding / Electroporation current: 8 step control • Frequency accuracy: within 1 %

• • • •

Complex impedance Z, R and C Well capacity: 16 or 96 wells Ability to model multi-frequency data (barrier function) Maximum data acquisition rate (single frequency): 5 point / sec • Multi-frequency measurements: 11 frequencies ranging from 50 Hz to 100 kHz (10 seconds per well acquisition rate) • Wounding / Electroporation current: 256 step control • Frequency accuracy: within 0.002 %

Impedance-Based Cell Assays

Dimensions: 33 x 11 x 33 cm Weight: 2.7 kg

General Specifications for ECIS Z and ECIS Zθ 16 Well Array Station

96 Well Array Station

• Dimensions: 13 x 15 x 4 cm

• Dimensions: 18 x 25 x 6 cm

71612

16 well station, which contains: 2 slots for ECIS Cultureware 8 well array holders, and a data processing station

71614

96 well station, which contains: A 96 well holder and a data processing station

Flow Option

Wound / Electroporate Option

• Ability to measure under dynamic flow conditions (Pump System, see page 118)

• User specified wound time, current and frequency • Delayed wounding during data collection

71001

ECIS Flow Module perfusion system for ECIS flow arrays, ideal for defined flow rates and shear stress studies of endothelial cells

Functionality already included in both ECIS systems

Operating Systems: Windows 7, Vista, XP, Mac OSX (10.5)

143


in cooperation with

Applied BioPhysics

www.biophysics.com

ECIS Cultureware The ECIS Cultureware consists of sterile disposable electrode arrays containing gold film electrodes. The gold layer is thin enough to allow microscopic observation of the cells using a standard inverted tissue culture microscope. The ECIS electrode array is placed in an array holder located in the incubator.

Substrates for ECIS Cultureware

Impedance-Based Cell Assays

Applied Biophysics offers two different substrates for the ECIS cultureware. The classic Lexan substrate offers superb imaging quality through the thin gold layer. It gives best results if ECIS measurements are combined with microscopy. In 2013, Applied Biophysics has developed a substrate, made out of PET, for all arrays. In these arrays, the gold electrode is located on 254 µm thick PET material and is optically clear, so that cells can be viewed on an inverted microscope. This base material has become the new standard. With the PET substrate, Applied Biophysics is offering an economic alternative to the already existing Lexan substrates. If you require Lexan arrays, please contact us at info@ibidi.de.

Lexan Substrate Gold Electrode

Cells Insulating Film

PET Substrate

Cells Insulating Film

Gold Electrode

Types of ECIS Cultureware

8W1E Each of the 8 wells contains a single circular 250 µm diameter measuring electrode*. Applications include: Cat. No. Descr. 72001

8W1E PET

Pcs. / Box 6

• Cell migration measurements via automated wound healing • Studying cell motion (i.e., micromotion) • Studies of very sparse cultures, even single cells

8W10E Each of the 8 wells contains ten 250 µm diameter measuring electrodes connected in parallel on a common gold pad*. Applications include: Cat. No. Descr. 72010

8W10E PET

Pcs. / Box 6

• Recording the activities of more cells over a larger area of the substrate • Studying the effects of agents upon overall impedance • Reducing fluctuations in impedance due to micromotion

8W10E+ Each of the 8 wells contains forty 250 µm diameter measuring electrodes connected in parallel on a common gold pad*. The array records the activities of more cells over a larger area of the substrate. Cat. No. Descr. 72040

144

8W10E+ PET

Pcs. / Box 6

Applications include: • • • •

Cell-ECM protein interactions Invasion detection of endothelial cell layers by metastatic cells Signal transduction assays Barrier function measurements


8W1E DD This array is nearly identical to the 8W1E standard array but the active electrodes* come in four different diameters (two wells of each size). The diameters are 250 (standard), 100, 50 and 25 µm. Application: • Monitoring fewer cells with higher sensitivity

Cat. No. Descr. 72003

Pcs. / Box

8W1E DD PET

6

8W2x1E (Medusa)

Applications include: • Wound healing studies • Migration • Electroporation

Cat. No. Descr. 72002

Pcs. / Box

8W2x1E (Medusa) PET

6

8WCP Each of the 8 wells contains two sets of four 7.5 mm x 0.5 mm rectangular electrodes located on inter-digitated fingers so as to provide cell measurements upon a total of 8 large electrodes. Application: • Cell proliferation assays

Cat. No. Descr. 72030

8WCP PET

Pcs. / Box 6

8W2LE

Impedance-Based Cell Assays

This array contains 8 individual wells, with each well having two independent standard measuring electrodes*. This allows to perform duplicate measurements within a single well.

Each of the 8 wells contains a single 667 µm x 150 µm linear electrode whose area is equal to that of our standard 250 µm circular electrodes. Applications include: • Cell migration • Wound healing assays

Cat. No. Descr. 72005

8W2LE PET

Pcs. / Box 6

8W1LE ChemoTaxis This array is used in chemotaxis measurements as first described by Hadjout, N. et al. in Biotechniques, 31 (5): 1130-1138 (2001). The measuring electrode in this array is a thin gold line between two registry marks*. Cat. No. Descr. 72021

8W1LE PET

Pcs. / Box 6

145


ECIS Cultureware Types of ECIS Cultureware

96W1E+ | 96W10idf | 96W20idf

N

Each of the 96 wells in a standard plate configuration contains two circular 350 µm diameter measuring electrodes* (96W1E+) or has an inter-digitated finger configuration (96W10idf, 96W20idf). These arrays have been designed for high throughput assays. The exceptional signal-to-noise ratio allows studies of very sparse cultures, even single cells.

Cat. No. Descr.

Pcs. / Box

72096

96W1E+PET

6

72097

96W10idf PET

6

72098

96W20idf PET

6

• Signal transduction assays • Barrier function measurement • Cell migration and proliferation

ECIS Flow Array 1E | 10E A specialized array with 8 active ECIS electrodes* located in the central region at the base of a flow channel (50 mm length, 5 mm width, around 0.35 mm height). Flow characteristics are comparable to the µ-Slide I 0.4 Luer (see p. 128).

N

Applications include:

EW

Impedance-Based Cell Assays

EW

Applications include:

Cat. No. Descr.

Pcs. / Box

70101

ECIS Flow Array 1E

6

70110

ECIS Flow Array 10E

6

• Studying cells under defined shear stress • Formation of tight junctions like the brain blood barrier

ECIS Transwell Measurement System N

Designed to measure the TEER (Trans Epithelial / Endothelial Electric Resistance) of cell layers grown on filter inserts

EW Cat. No. Descr.

• Easily comparable to classic TEER measurements

71609

• Ideal for addressing lung epithelial cell cultures

ECIS Transwell Measurement System

The ECIS Transwell Measurement System is a permanent transwell holder to measure the TEER of cell layers. The holder contains a lower Filter holder base and an upper counter electrode system. It is used in filter-based assays.

146

* Gold films are transparent and the upper chambers can be removed after ECIS measurements to facilitate immunofluorescence staining. For details, please refer to the microscopy guide on www.biophysics.com.


Âľ-Slide I Luer electrode . . . . . . . . . . . . . . . . . . . . 148

Custom Specific Slides

Custom Specific Flow Slides and Channels . . . . . . . . . . . . . . . . . . . . . . . . . 149

147


µ-Slide I Luer electrode Designed for simultaneous microscopic and electric analysis of cells

• Customizable electrode layouts • Compatible with the ibidi Heating System

Applications • Applying and measuring the defined voltage of electric fields • Electrical stimulation of cells (e.g., heart muscle cells) • Electrotaxis (i.e., a directed cell migration in an electric field) • Polarization of cells by electric fields

Custom Specific Slides

• Conducting apoptosis and necrosis assays by measuring resistance changes over the channel

Available on request:

Technical Features:

• Contact module with integrated amplifier

• Resistance of approx. 300 Ohm • Electrodes on a #1.5 glass support

• Insulated tracks • Custom-specific electrode arrangements

• Electrodes based on Ti-TiN layer • Available with different channel heights ranging from 0.1 mm to 0.8 mm • Contact module with 8 to 32 ports

Contact Module Compatible with the ibidi Heating System

Electrode Layout

Contact module upper part

Electrode slide

Contact module lower part

Ordering Information:

148

Cat. No.

Description

82000

Custom specific µ-Slide I Luer electrode

10939

Contact Module

Pcs. / Box 15 1


Custom Specific Flow Slides and Channels µ-Slides with custom-specific channel geometry and height

• Develop your own geometry!

Developed Formats

• If you are interested in a custom specific solution please contact us directly at customer.solutions@ibidi.com.

Applications

µ-Slide III 0.4 (see page 131)

• Create channels with your own particular characteristics • Use as a microfluidic workbench • Cell culture flow applications

Technical Features

Custom Specific Slides

µ-Slide III 0.1 (see page 130)

• Format and material based on µ-Slides • Additional materials possible, like glass coverslips, silicone designs, permeable membranes, or electrodes • Channel height and widths available in different dimensions • 2 x 3 Luer adapters in fixed positions

µ-Slide III 3in1 (see page 111)

Several Possible Variations

Branching channels

Cross channels

Straight channels

Meander channels

Ordering Information: Cat. No.

Description

Please contact us for further details at customer.solutions@ibidi.com

Specifications: Pcs. / Box

Adapters

Female Luer

Volume per reservoir

60 µl

Height of channels

0.05 - 0.6 mm

Width of channels

0.1 - 10 mm

149


Custom Specific Slides

Develop Your Own Slide Format

150


WimComet – Comet Assay Image Analysis . . . . . . . . . . . . 160

WimSprout – Sprouting Image Analysis . . . . . . . . . . . . . . . 153

WimColony – Colony-Forming Image Analysis . . . . . . . . . . 161

WimTaxis – Chemotaxis Image Analysis . . . . . . . . . . . . . 154

WimNeuron – Neurite Outgrowth Image Analysis . . . . . . . . 162

Chemotaxis and Migration Tool . . . . . . . . . . . 155

WimRetina – Retina Vessel Image Analysis . . . . . . . . . . . . 163

WimScratch – Wound Healing Image Analysis . . . . . . . . . . . 156 WimCounting – Cell Counting Image Analysis . . . . . . . . . . . . 157 WimCAM – Chorioallantoic Membrane Assay Image Analysis . . . . . . . . . . . . . . . . . . . . . . . 158 WimCytotoxicity – Cytotoxicity Image Analysis . . . . . . . . . . . . . 159

WimTransfection – Transfection Efficiency Image Analysis . . . . . 164 WimLipid – Lipid Droplets Image Analysis . . . . . . . . . . . . 165 WimAdipose – Adipose Tissue Image Analysis . . . . . . . . . . 166 WimTUNEL – TUNEL Assay Image Analysis . . . . . . . . . . . 167

Image Analysis

WimTube – Tube Formation Image Analysis . . . . . . . . . . 152

151


WimTube – Tube Formation Image Analysis

• Complete solution for tube formation experiments – only a few steps from sample preparation to image analysis

8000

6000

• Objective and reproducible analysis

4000

• Easy and fast data processing – results within minutes

2000

• No extra hardware or software needed

0

1

Co nt ro l ng /m l 10 ng /m l 10 0 ng /m l 1 µg /m l 10 µg /m l

Tube length [µm*]

* Lenght in µm / Standard Area (=1 cm²)

A web-based quantitative image analysis of tube formation assays for the investigation of angiogenesis

Concentration

Inhibition of tube formation by a compound in a dose – effect relation

Applications • Quantification of tube formation assays • Image analysis and data interpretation of angiogenesis experiments • Investigation of tube formation inhibition

ibidi TIP

Image Analysis

Use our Free Trial at www.ibidi.com to test the WimTube Image Analysis for yourself.

Tube Formation Image Analysis is a crucial step in angiogenesis assays. Wimasis, the certified partner of ibidi for quantitative image analysis, developed an analysis solution to quantitatively evaluate the generation of cellular networks. First, use the µ-Slide Angiogenesis or the µ-Plate Angiogenesis 96 well (see pages 100  /  101) to perform your tube formation experiments and acquire microscopy images. Then, simply upload the images to the image analysis platform and receive a summary of results, via E-mail, and download the detailed results.

ibidi MOVIE Learn more about WimTube in the movie,

Analysis Data Contains:

Tube Formation Assay Evaluated with the Wimasis Automated Image Analysis (MV 23), on www.ibidi.com.

• Tube lengths, numbers, and mean values • Cell-covered area • Loop numbers, areas, and perimeters • Branching points

Only 4 Steps From Sample Preparation to Image Analysis • Branching points • Tube lengths, numbers, and statistics • Loop numbers, areas, and perimeters • Cell-covered area

Gel matrix

1. Cell seeding and tube formation

2. Acquisition of microscopy images

4. Data analysis and evaluation

in cooperation with

Ordering Information:

152

3. Quantitative image analysis

Cat. No.

Description

3000150 / 100 / 200 / 500 / 1000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimTube Quantitative Tube Formation Image Analysis, 50 / 100 / 200 / 200 / 500 / 1,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images


WimSprout – Sprouting Image Analysis N

EW

A web-based, quantitative image analysis of sprouting assays for the investigation of angiogenesis and tumor growth

1500 1000 500 0

C

on

• Available in three different modules: Sprouting Spheroid Assay, Aortic Ring Assay and Fibrin Gel Bead Assay

***

+ VE su G bs F t. 1 + VE su G bs F t. 2

• Easy and fast data processing – results within minutes

***

2000

tro l

• Objective and reproducible analysis

2500

VE G F

Cumulative Sprout Length in µm

• Complete solution for sprouting assays in angiogenesis research – only a few steps from sample preparation to image analysis

Applications • Quantification of sprouting assays • Image analysis and data interpretation of aortic ring assays • Investigation of sprouting enhancement and inhibition

ibidi TIP

• Fibrin Gel Bead Assays

First, use the µ-Slide Angiogenesis or the µ-Plate Angiogenesis 96 well (see pages 100 / 101) as a suitable platform for gel application and the performance of your sprouting assay. Next, acquire microscopy images and simply upload the images to the image analysis platform. Finally, you will receive a summary of results, via E-mail, in under an hour. You can then download your detailed results.

Analysis Data Contains: • Spheroid/aortic ring area • Sprouts area • Number of sprouts • Cumulative sprouts length

1500 1000 500 0

C

on

Sprouting

***

1. Sprouting spheroid

2. Acquisition of microscopy images

3. Quantitative image analysis

+ VE su G bs F t. 1 + VE su G bs F t. 2

Cell spheroid

***

2000

VE G F

Gel matrix

2500

tro l

Cumulative Sprout Length in µm

Only 4 Steps From Sample Preparation to Image Analysis

Automated Image Analysis

The image analysis of sprouting spheroids or aortic rings is a crucial step in sprouting assays. Wimasis, the certified partner of ibidi for quantitative image analysis, has developed an analysis solution to quantitatively evaluate the building of sprouts in a gel matrix. There are three different modules, adapted for sprouting spheroids, aortic ring-like structures, and fibrin gel bead assays, respectively.

Use our Free Trial at www.ibidi.com to test the WimSprout Image Analysis for yourself.

4. Data analysis and evaluation

in cooperation with

Ordering Information: Cat. No.

Description

3000450 / 100 / 200 / 500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimSprout Quantitative Sprouting Spheroid Assay Image Analysis,

3001450 / 100 / 200 / 500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimSprout Quantitative Aortic Ring Assay Image Analysis,

3002450 / 100 / 200 / 500 / 1000

WimSprout Quantitative Fibrin Gel Bead Assay Image Analysis,

50 / 100 / 200 / 500 / 1,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images

50 / 100 / 200 / 500 / 1,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images

50 / 100 / 200 / 500 / 1,000 images

153


WimTaxis – Chemotaxis Image Analysis A web-based, quantitative image analysis of chemotaxis assays with automated tracking of label-free cell lines and bacteria in phase contrast

• Complete solution for chemotaxis experiments – only a few steps from sample preparation to image analysis

y displacement [µm]

200

• Objective and reproducible analysis

100

• Easy and fast data processing – results within hours 0

• Available in the modules Cell Tracking and Bacteria Tracking

-100

Applications

-200 -200

-100

0

100

200

x displacement [µm]

• Tracking of cells or particles in time lapse records • Image analysis and quantification of 2D chemotaxis assays

Trajectory plot

• Quantification and data interpretation of bacterial chemotaxis

Image Analysis

Analysis Data Contains: • Cell tracking positions with animated trajectory plot • Forward migration index (parallel and perpendicular) • Cell video with tracks • Track and center of mass statistics For further analysis, data can be imported into ibidi’s Chemotaxis and Migration Tool (see page 155).

ibidi TIP Test your own data first, with a Free Trial at www.ibidi.com, to make sure that your image quality is sufficient for this type of analysis.

Chemotaxis Image Analysis is a crucial aspect in many oncologyrelated research areas. Manual acquisition and evaluation of cell migration is time consuming and lacks in objectivity. Wimasis, the certified partner of ibidi for quantitative image analysis, developed an analysis solution that quantitatively evaluates chemotaxis and migration. Because there are differences between the migration characteristics of adherent cells and bacteria, there are special modules for adherent cell and bacterial chemotaxis analysis. First, perform your cellular chemotaxis experiments, using either the µ-Slide Chemotaxis 2D or µ-Slide Chemotaxis 3D (see page 109) and record a time lapse movie. For bacterial chemotaxis, the protocol must be adapted accordingly. Next, simply upload your data to the image analysis platform. WimTaxis makes an accurate detection of both cell and bacteria movement in chemotaxis assays. It tracks cells and bacteria and provides their trajectories throughout the video. Just choose the WimTaxis that best fits your assay needs, and then get your analysis done fast and consistently.

Only 4 Steps From Sample Preparation to Image Analysis 200

p-value (Rayleigh-Test)

20 min 10 0 10 10 10

-4 -8

100

0

0.3

-100 FMI

10 min

y displacement [µm]

0 min

-12

-200 +/-

-/-

-200

0.2 0.1 0.0

+/+

-100

+/-

0

-/-

100 +/+

x displacement [µm]

1. Cell seeding and chemotaxis assay

2. Acquisition of microscopy images

3. Automated cell tracking

in cooperation with

Ordering Information:

154

4. Data analysis and evaluation

Cat. No.

Description

3000310 / 50 / 100 / 200 / 500 / 1000

WimTaxis Quantitative Chemotaxis Image Analysis, 10 / 50 / 100 / 200 / 200 / 500 / 1,000 videos One video contains 250 images maximum.

200


Chemotaxis and Migration Tool A free software tool for data analysis from time stack chemotaxis experiments, based on the National Institute of Health’s (NIH) ImageJ image processing system

• A free and platform independent solution • Provides various graphs and statistical tests that allow advanced analysis of chemotaxis experiments

Applications • Image analysis and data interpretation of chemotaxis and migration time lapse movies • Quantification of 2D and 3D chemotaxis time lapse movies

The Chemotaxis and Migration Tool is freely available on www.ibidi.com. The tool provides various graphs and statistical tests that allow advanced analysis of experimental data.

• Trajectory plots • Migration characteristics (COM, FMI, Directness, Velocity) • Statistical tests and detailed results per track or per slice

Optionally, all data can be exported for further analyses. With a complete set of migration data, the user is able to quantify chemotaxis and random migration. The Chemotaxis and Migration Tool, together with the µ-Slide Chemotaxis 2D or the µ-Slide Chemotaxis 3D (see page 109) enables users to analyze in minute detail the effect of drugs on the chemotactic behavior of cells. Example Data

CT-Exp. (+/-)

Control 1 (-/-)

Control 2 (+/+)

Center of mass x [µm]

-4.0

3.6

-7.6

Center of mass y [µm] FMI perpendicular (  _I )

176.3

18.1

-3.1

-0.014

0.015

-0.004

FMI parallel (II)

0.280

0.035

-0.007

Directness

0.33

0.16

0.21

Mean cell velocity [µm / min]

0.43

0.29

0.49

< 0.05

> 0.05

> 0.05

Rayleigh test (p value)

ibidi TIP For a free download of the Chemotaxis and Migration Tool please go to www.ibidi.com. For manually tracking cells in a time stack, ibidi recommends using the Manual Tracking plug in. This plug in is available for download on: http://rsb.info.nih. gov/ij/plugins/track/track.html

Automated Image Analysis

After cell tracking, the cells‘ paths can be plotted and analyzed for chemotactical effects. The simple to use microscopic calibration system provides access to a variety of parameters. A set of statistical tests complete the software package.

Data Evaluation Contains:

155


WimScratch – Wound Healing Image Analysis A web-based quantitative image analysis of wound healing and cell migration assays

• A complete solution for wound healing experiments – only a few steps from sample preparation to image analysis

Cell Front Velocity [µm/h]

35 30 25

• Objective and reproducible analysis

20 15

• Easy and fast data processing – results within minutes

10

• No extra hardware or software needed

5

Co nt ro In hi l bi to In r1 hi bi En tor 2 ha n En cer 1 ha nc er 2

0

Statistical analysis of a complete experimental series

Applications • Quantification of wound healing and cell migration assays • Characterization of wound closure time lapse movies • Image analysis and data interpretation of migration experiments

ibidi TIP

Image Analysis

Use our Free Trial at www.ibidi.com to test the WimScratch Image Analysis for yourself.

The Wound Healing Image Analysis solution evaluates 2D cell migration. Use the ibidi Culture-Inserts (see page 94) to do your migration experiments. After acquiring the microscopy images, simply upload your data to the image analysis platform. Receive a summary of results, via E-mail, and download the detailed results. Analysis Data Contains:

ibidi MOVIE

• Cell-covered area

Learn more about WimScratch in the movie,

• Speed of closure (available ≥ 5 images)

Wound Healing Assay Evaluated with the Wimasis Automated Image Analysis (MV 22),

• Acceleration characteristics (available ≥ 5 images) • Overview chart • Center piece approximation (available ≥ 10 images)

on www.ibidi.com.

Only 4 Steps From Sample Preparation to Image Analysis Cell covered area [%]

100 95 90 85 80 75 0

20

• Cell covered area • Speed of closure • Acceleration characteristics • Overview chart 40

60

80

100

Time [h]

1. Cell seeding and gap formation

2. Acquisition of microscopy images

4. Data analysis and evaluation

in cooperation with

Ordering Information:

156

3. Quantitative image analysis

Cat. No.

Description

30002200 / 500 / 1000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimScratch Quantitative Wound Healing Image Analysis, 200 / 500 / 1,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images


WimCounting – Cell Counting Image Analysis N

EW

A web-based, quantitative image analysis for cell or particle counting

• Automated counting of cells in solution (rounded-up) • Objective and reproducible analysis • Easy and fast data processing – results within minutes • No more manual cell counting

Applications • Cell counting of detached, rounded-up cells (e.g., in tube formation assays) • Particle counting • Counting of fluorescence-labeled cells

ibidi TIP

Wimasis, the certified partner of ibidi for quantitative image analysis, has developed an analysis solution to automatically detect and count cells. The Image Analysis solution generates fast results, without the need for extra hardware or software. Detach your cells and bring them into one focal plane (e.g., by filling them into a counting chamber). You can also wait 15-30 minutes until all cells have fallen to the bottom of the culture vessel. Next, take a picture of the rounded-up cells and upload your data. Finally, you will receive a summary of results, via E-mail, in under an hour. You can then download your detailed results. Analysis Data Contains: • Cell number • Images with highlighted counted cells

For cell culture ibidi recommends the µ-Slide 2 well | 4 well | 8 well or the µ-Dish 35 mm, high (see page 66 and 61).

ibidi TIP Use our Free Trial at www.ibidi.com to test the WimCounting Image Analysis for yourself.

Automated Image Analysis

Cell counting is a crucial step in many experimental protocols. It assures the correct starting conditions. Manual cell counting is tedious and time consuming.

in cooperation with

Ordering Information: Cat. No.

Description

30005200 / 500 / 1000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimCounting Quantitative Cell Counting Image Analysis, 200 / 500 / 1,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images

157


WimCAM – Chorioallantoic Membrane Assay Image Analysis N EW

A web-based quantitative image analysis of chorioallantoic membrane assays with automated detection of blood vessel formation

• Complete solution for chorioallantoic membrane assays – only a few steps from sample preparation to image analysis • Objective and reproducible analysis • Easy and fast data processing - results within hours • No extra hardware or software needed

Image Analysis

Image granted by the MRC Centre for Developmental and Biomedical Genetics, University of Sheffield and Dept. of Cardiovascular Sciences, University of Leicester

Applications • Quantification of neovascularization and angiogenesis studies • Quantification of drug effects on blood vessel formation • Screening and biocompatibility tests in chorioallantoic membrane assays

WimCAM is the image analysis solution that was specially developed to objectively quantify the growth and structure of the newly generated vessels in the chorioallantoic membrane. It provides biological and biomedical researchers with objective and reliable measurements of the vascular structure, which then enables the objective comparison of the results.

ibidi TIP Use our Free Trial at www.ibidi.com to test the WimCAM Image Analysis for yourself.

First, perform your chorioallantoic membrane assays and image the vessel formation. Then, simply upload your data to the image analysis platform, receive a summary of results, and download the detailed results. Analysis Data Contains: • Number of vessel segments • Number of branching points • Number of nets

• Vessel density • Mean segment length • Total vessel network length

Detailed Description of the Analysis: Because every experimental setup is unique, the WimCAM tool is engineered with the flexibility to adapt to the needs of every researcher. If your image data does not fit the requirements above, send us a quick note at info@ibidi.de, so that we can find a solution that fits your experiment.

For the input data, WimCAM uses bright field microscopy images of the chorioallantoic membrane of chicken eggs. Images taken of the inside of the egg and of a piece of eggshell are both suitable.

in cooperation with

Ordering Information:

158

Cat. No.

Description

30006500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimCAM Quantitative Chorioallantoic Membrane Assay Image Analysis, 500 / 1,000 / 2,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images


WimCytotoxicity – Cytotoxicity Image Analysis N

EW

A web-based quantitative image analysis of cytotoxicity assays with automated detection of live and affected/dead cells

• Complete solution for cytotoxicity assays – only a few steps from sample preparation to image analysis • Objective and reproducible analysis • Easy and fast data processing - results within hours • No more manual cell counting

Applications • Quantification of cytotoxicity assays • Quantification of live-dead stainings • Image analysis and data interpretation of vitality tests

First, perform your cytotoxicity experiments, with a subsequent fluorescence staining. Then, simply upload your data to the image analysis platform, receive a summary of results, and download the detailed results. ibidi TIP

Analysis Data Contains: • Total cell count: number of all visible cells in the culture • Number of affected / dead cells stained by the cytotoxicity fluorescence marker • Viability of cells (percentage of affected/dead cells of the whole population)

Use our Free Trial at www.ibidi.com to test the WimCytotoxicity Image Analysis for yourself.

Detailed Description of the Analysis: For the input data, WimCytotoxicity uses fluorescence microscopy images with two different dyes: one dye for the viable cell population and another one for the cytotoxicity-affected cells. Optionally, an extra nuclear dye can be applied to stain cellular nuclei, which will then improve the accuracy of the cell detection algorithm. The quantification is based on the detection of the whole cell population and the identification of the cells affected by the cytotoxic agents.

Because every experimental setup is unique, the WimCytotoxicity tool is engineered with the flexibility to adapt to the needs of every researcher. If your cytotoxicity assay does not fit the requirements above, send us a quick note at info@ibidi.de, so that we can find a solution that fits your experiment.

Automated Image Analysis

The use of fluorescent dyes to assess cellular viability under the effect of cytotoxic agents is an extended practice in pharmaceutical research. It is a key assay in screening the cytotoxic potential of new drug compounds, as well as the study of cell-mediated cytotoxicity, or the evaluation of the effects of cytotoxic pollutants in cells.

in cooperation with

Ordering Information: Cat. No.

Description

30007500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimCytotoxicity Quantitative Cytotoxicity Image Analysis, 500 / 1,000 / 2,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images

159


WimComet – Comet Assay Image Analysis N EW

A web-based quantitative image analysis of comet assays with automated readout of fluorescence intensities

• Complete solution for comet assay readout – only a few steps from sample preparation to image analysis • Objective and reproducible analysis • Easy and fast data processing - results within hours • No extra hardware or software needed

Applications • Detection of DNA damage in comet assays (single-cell gel electrophoresis, SCGE) • Studies of genotoxicity • Sperm DNA fragmentation

Image Analysis

WimComet is an image analysis solution that is specially designed to objectively measure the DNA damage in comet assay images, with high precision and reliability. It provides an automated and reproducible quantification of the comet head and tail by using highend image processing algorithms to detect and compare the signals between the two of them.

ibidi TIP Use our Free Trial at www.ibidi.com to test the WimComet Image Analysis for yourself.

First, perform your single cell gel electrophoresis and record your fluorescence images. Then, simply upload your data to the image analysis platform, receive a summary of results, and download the detailed results. Analysis Data Contains: • Total fluorescence intensity of each single cell • Comet length • Percentage of DNA in the tail • Tail length

Because every experimental setup is unique, the WimComet tool is engineered with the flexibility to adapt to the needs of every researcher. If your image data does not fit the requirements above, send us a quick note at info@ibidi.de, so that we can find a solution that fits your experiment.

• Olive tail moment (tail length multiplied by the percentage of DNA in the tail) Detailed Description of the Analysis: For the imput data, WimComet uses fluorescence microscopy images of the comet assay, where the bright comets are shown in a dark background. Both images, with one or several comets, can be analyzed.

in cooperation with

Ordering Information:

160

Cat. No.

Description

30008500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimComet Quantitative Comet Assay Image Analysis, 500 / 1,000 / 2,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images


WimColony – Colony-Forming Image Analysis N

EW

A web-based, quantitative image analysis of colony-forming processes with automated detection of cell colonies

• Complete solution for colony-forming assays - only a few steps from sample preparation to image analysis • Objective and reproducible analysis • Easy and fast data processing - results within hours • No extra hardware or software needed

Applications • Quantification of colony formation and proliferation processes • Quantification of soft agar assays (clonogenic assays)

First, perform your colony-forming assays and image the colony building and transformation. Then, simply upload your data to the image analysis platform, receive a summary of results, and download the detailed results. ibidi TIP

Analysis Data Contains: • Number of detected colonies / objects • Classification of colonies into size categories • Total area of colony / object surface • Mean colony/object area

Use our Free Trial at www.ibidi.com to test the WimColony Image Analysis for yourself.

Detailed Description of the Analysis: For the input data, WimColony uses bright field microscopy images of the colony formation. Images of bacterial and eukaryotic cell colony formation are both suitable.

Because every experimental setup is unique, the WimColony tool is engineered with the flexibility to adapt to the needs of every researcher. If your image data does not fit the requirements above, send us a quick note at info@ibidi.de, so that we can find a solution that fits your experiment.

Automated Image Analysis

WimColony is an image analysis solution that was specially developed to track the progress of colony formation in several cellbased assays (e.g., soft agar assay, clonogenic assay). It provides biological and biomedical researchers with objective and reliable measurements of the colony formation characteristics, which then allows for an objective comparison of the results.

in cooperation with

Ordering Information: Cat. No.

Description

30009500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimColony Quantitative Colony Forming Image Analysis, 500 / 1,000 / 2,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images

161


WimNeuron – Neurite Outgrowth Image Analysis N EW

A web-based, quantitative image analysis of neurite outgrowth with automated detection of cell soma and neurites

• Complete solution for monitoring neurite outgrowth - only a few steps from sample preparation to image analysis • Objective and reproducible analysis • Easy and fast data processing - results within hours • No extra hardware or software needed

Applications Phase contrast images of neurons in cell culture

• Monitoring the neurite outgrowth process • Quantification of the neurite growth induction • Screening for neurotoxicity of compounds

Image Analysis

WimNeuron is an image analysis solution that was specially developed to objectively quantify the outgrowth of neurites from a cell body. It provides biological and biomedical researchers with objective and reliable measurements of the neurite structure, which then allows for an objective comparison of the results. First, perform your Neurite Outgrowth Assay and image the formation of projections. Next, simply upload your data to the image analysis platform. Finally, receive a summary of results, via E-mail, and download the detailed results. ibidi TIP Use our Free Trial at www.ibidi.com to test the WimNeuron Image Analysis for yourself.

Analysis Data Contains: • Number of neuronal networks • Number of neurite segments • Number of branching points • Average neurite length • Area covered by neurites/cell soma • Total neurite network length

Because every experimental setup is unique, the WimNeuron tool is engineered with the flexibility to adapt to the needs of every researcher. If your image data does not fit the requirements above, send us a quick note at info@ibidi.de, so that we can find a solution that fits your experiment.

Detailed Description of the Analysis: For the input data, WimNeuron uses phase contrast, bright field or fluorescence microscopy images of the neurite network. Optionally, neurons can be stained with nuclear dyes (such as DAPI or Hoechst), which will allow for the provision of some extra parameters of the analysis.

in cooperation with

Ordering Information:

162

Cat. No.

Description

30010500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimNeuron Quantitative Neurite Outgrowth Image Analysis, 500 / 1,000 / 2,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images


WimRetina – Retina Vessel Image Analysis N

EW

A web-based, quantitative image analysis of retina vessels with automated detection of blood vessel formation

• Complete solution for retina vessel investigations - only a few steps from sample preparation to image analysis • Objective and reproducible analysis • Easy and fast data processing - results within hours • No extra hardware or software needed

Applications • Quantification of blood vessel growth and development • Characterization of the blood vessel structure • Screening of angiogenesis-inhibiting compounds

Fluorescence image of the vessels of a retina petal (image permission granted by the Centenary Institute of Cancer Medicine and Cell Biology, Australia).

First, perform your retina vessel assay and image the vessel formation. Next, simply upload your data to the image analysis platform. Finally, receive a summary of results, via E-mail, and download the detailed results. Analysis Data Contains: • Number of vessel segments

• Vessel density

• Number of branching points

• Mean segment length

• Number of nets

• Total vessel network length

Detailed Description of the Analysis: For the input data, WimRetina uses fluorescence microscopy images of the retina with a clear contrast between the dark background and the fluorescence-labeled vessels. Images of the whole retina (flower-like structure), or sections of only a part of the vasculature, are both suitable for the analysis.

ibidi TIP Use our Free Trial at www.ibidi.com to test the WimRetina Image Analysis for yourself.

Because every experimental setup is unique, the WimRetina tool is engineered with the flexibility to adapt to the needs of every researcher. If your image data does not fit the requirements above, send us a quick note at info@ibidi.de, so that we can find a solution that fits your experiment.

Automated Image Analysis

WimRetina is an image analysis solution that was specially developed to objectively quantify the growth and structure of the newly generated vessels in the retina. It provides biological and biomedical researchers with objective and reliable measurements of the vascular structure, which then allows for an objective comparison of the results.

in cooperation with

Ordering Information: Cat. No.

Description

30101500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimRetina Quantitative Retina Vessels Image Analysis, 500 / 1,000 / 2,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images

163


WimTransfection – Transfection Efficiency Image Analysis N EW

A web-based, quantitative image analysis of transfection efficiency with automated detection of transfected cells

• Complete solution for transfection efficiency evaluation only a few steps from sample preparation to image analysis • Objective and reproducible analysis • Easy and fast data processing - results within hours • No extra hardware or software needed

Application HepG2 cells transfected with Torpedo DNA (see page 50). Transfected cells are expressing GFP.

• Quantification of transfection efficiency

Image Analysis

WimTransfection is an image analysis solution that was specially developed to objectively quantify the growth and expression efficiency of transfected cells. It provides biological and biomedical researchers with objective and reliable measurements of the transfected cell culture, which then allows for an objective comparison of the results. First, perform your transfection protocol and image the layer of transfected cells. Next, simply upload your data to the image analysis platform. Finally, receive a summary of results, via E-mail, and download the detailed results. Analysis Data Contains: ibidi TIP Use our Free Trial at www.ibidi.com to test the WimTransfection Image Analysis for yourself.

Because every experimental setup is unique, the WimTransfection tool is engineered with the flexibility to adapt to the needs of every researcher. If your image data does not fit the requirements above, send us a quick note at info@ibidi.de, so that we can find a solution that fits your experiment.

• Total number of cells (total cell count) • Number of transfected cells • Transfection efficiency • Cell covered area Detailed Description of the Analysis: For the input data, WimTransfection uses fluorescence images of the transfected cells (fluorescence labeled) and phase contrast or fluorescence images that are visualizing the whole cell layer. One analysis consists of a pair of pictures, such as the phase contrast image of the cell layer and the overlaid fluorescence image.

in cooperation with

Ordering Information:

164

Cat. No.

Description

30102500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimTransfection Quantitative Transfection Efficiency Image Analysis, 500 / 1,000 / 2,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images


WimLipid – Lipid Droplets Image Analysis N

EW

A web-based, quantitative image analysis of lipid dropletscontaining cell images with automated detection and quantification of distribution and size of the droplets • Complete solution for lipid droplets investigation - only a few steps from sample preparation to image analysis • Objective and reproducible analysis • Easy and fast data processing - results within hours • No more manual image analysis or counting

Applications • Detection and quantification of lipid droplets in cells • Quantification of droplet distribution over time • Characterization of cells in metabolic diseases

Lipid Droplets images courtesy of the ZIEL Molecular Nutritional Medicine Department of the Technische Universität München.

First, perform your cell culture experiments and record your microscopy images. Then, simply upload your data to the image analysis platform. Finally, receive a summary of results, via E-mail, dan download the detailed results. Analysis Data Contains: ibidi TIP

• Total number of lipid droplets • Total area covered with lipid droplets • Mean size of lipid droplets • Distribution indicated as areas per interval

Use our Free Trial at www.ibidi.com to test the WimLipid Image Analysis for yourself.

Detailed Description of the Analysis: For the input data, WimLipid uses brightfield microscopy images with or without staining of the lipid bodies. The lipid droplets are visible as bright shining spots due to their refractile properties. Optionally, an extra nuclear dye can be applied to stain cellular nuclei, which will then improve the results, thus providing more information about the distribution of the droplets inside the cell. The quantification is based on the detection of the droplets and the related covered area. In addition, parameters, such as mean size and distribution, are calculated.

Because every experimental setup is unique, the WimLipid tool is engineered with the flexibility to adapt to the needs of every researcher. If your experimental results do not fit the requirements above, send us a quick note at info@ibidi.de, so that we can find a solution that works with your experiment.

Automated Image Analysis

Lipid droplets are cell organelles that specialize in the storage and supply of lipids within the metabolism. They are highly dynamic structures that are involved in many regulation processes, such as inflammatory responses, protection from lipotoxicity or atherosclerosis.

in cooperation with

Ordering Information: Cat. No.

Description

30104500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimLipid Quantitative Lipid Droplets Image Analysis, 500 / 1,000 / 2,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images

165


40

28 27

33 26

WimAdipose – Adipose Tissue Image Analysis

30

31 23

41

39

29

34

38 32 36

N

25

EW

24

37

35

42 43

A web-based, quantitative image analysis of adipose tissue microscopic images with automated detection of cell characteristics, such as outlines, number and size • Complete processing of adipose tissue histological section images – only a few steps from sample preparation to image analysis • Optimized algorithms for detection of the whole cell population • Objective and reproducible analysis with detailed results • Easy and fast data processing - results within hours • No more manual image analysis

Adipose Tissue images courtesy of the ZIEL Molecular Nutritional Medicine Department of the Technische Universität München.

Applications • Quantification of adipocyte characteristics • Quantification of hematoxylin and eosin stained tissue sections

Image Analysis

• Detection of cell outlines

The investigation of adipose tissue characteristics is a powerful tool regarding many metabolic diseases, like diabetes and obesity. Specifically, the cross-sectional size and diameter of a cell are directly related to metabolic dysfunctions.

ibidi TIP Use our Free Trial at www.ibidi.com to test the WimAdipose Image Analysis for yourself.

First, make the section of your probe with a subsequent hematoxylin and eosin staining. Then, simply upload your data to the image analysis platform. Finally, receive a summary of results, via E-mail, dan download the detailed results. Analysis Data Contains: • Total cell count: number of all visible cells in the section • Mean area of the whole cell population with standard deviation • Mean equivalent diameter of the cells with standard deviation

Because every experimental setup is unique, the WimAdipose tool is engineered with the flexibility to adapt to the needs of every researcher. If your experimental results do not fit the requirements above, send us a quick note at info@ibidi.de, so that we can find a solution that works with your experiment.

Detailed Description of the Analysis: For the input data, WimAdipose uses microscopy images of hematoxylin and eosin stained adipose tissue sections. All standard image formats (jpg, tiff, bmp, etc.) are suitable for the analysis. The quantification is based on the detection of the whole cell population and the identification of the cell outlines. In addition, the cell surface and diameter are measured and the mean values are calculated.

in cooperation with

Ordering Information:

166

Cat. No.

Description

30103500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimAdipose Quantitative Adipose Tissue Image Analysis, 500 / 1,000 / 2,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images


WimTUNEL – TUNEL Assay Image Analysis N

EW

A web-based, quantitative image analysis of TUNEL assays with automated detection of live and affected/dead cells

• Complete solution for TUNEL assays - only a few steps from sample preparation to image analysis • Objective and reproducible analysis • Easy and fast data processing - results within hours • No more manual cell counting

Applications • Quantification of TUNEL assays • Quantification of live-dead stainings • Image analysis and data interpretation of vitality tests

First, perform your cell culture experiment with a subsequent fluorescence TUNEL staining. Then, simply upload your data to the image analysis platform. Finally, receive a summary of results, via E-mail, dan download the detailed results. Analysis Data Contains:

ibidi TIP

• Total cell count: number of all visible cells in the culture • Number of affected / dead cells stained by the TUNEL fluorescence marker • Viability of cells (percentage of affected/dead cells of the whole population)

Use our Free Trial at www.ibidi.com to test the WimTUNEL Image Analysis for yourself.

Detailed Description of the Analysis: For the input data, WimTUNEL uses fluorescence microscopy images with two different dyes: one dye for the apoptotic cell population and another for the whole cell population (live and apoptotic cells). Optionally, if a cytoplasmic membrane marker was used for the cell detection, an extra nuclear dye can be applied to stain cellular nuclei, which will then improve the accuracy of the cell detection algorithm. The quantification is based on the detection of the whole cell population and the identification of the apoptotic cells.

Because every experimental setup is unique, the WimTUNEL tool is engineered with the flexibility to adapt to the needs of every researcher. If you experimental results do not fit the requirements above, send us a quick note at info@ibidi.de, so that we can find a solution that works with your experiment.

Automated Image Analysis

Apoptosis is the programmed cell death that occurs, for example, in the ontogenesis of an organism or during normal regeneration processes, and in many pathological processes. Thus, it plays a key role in medical and pharmaceutical research. The TUNEL assay provides a tool that distiguishes necrotic from apoptotic cells.

in cooperation with

Ordering Information: Cat. No.

Description

30105500 / 1000 / 2000 / 5000 / 10000 / 20000 / 50000 / 1000000

WimTUNEL Quantitative TUNEL Assay Image Analysis, 500 / 1,000 / 2,000 / 5,000 / 10,000 / 20,000 / 50,000 / 100,000 images

167


Customer Feedback

“The ibidi cell migration Culture-Inserts enabled me to perform highly reproducible and quantitative migration experiments. Since physical scratching isn’t required, uniformity is maintained and adverse wounding responses are not an issue. The online quantification tool WimScratch for the assay is also fantastic and it yields quantitative data from the images extremely quickly with little effort required. I’d definitely recommend it for migration studies.“

Image Analysis

Ross Flockhart, PhD Stanford School of Medicine Department of Dermatology Stanford USA

References Regulation of ovarian cancer progression by microRNA-187 through targeting Disabled homolog-2 A. Chao, C. Y. Lin, Y. S. Lee, C. L. Tsai, P. C. Wei, S. Hsueh, T. I. Wu, C. N. Tsai, C. J. Wang and A. S. Chao Oncogene, 2011, 10.1038/ onc.2011.269

168

Tyrosine-phosphorylated Galectin-3 Protein Is Resistant to Prostate-specific Antigen (PSA) Cleavage V. Balan, P. Nangia-Makker, D. H. Kho, Y. Wang and A. Raz J. Biol. Chem., 2012, 10.1074/jbc. C111.331686

Heparin Strongly Induces Soluble fms-Like Tyrosine Kinase 1 Release In Vivo and In Vitro J. Searle, M. Mockel, S. Gwosc, S. A. Datwyler, F. Qadri, G. I. Albert, F. Holert, A. Isbruch, L. Klug and D. N. Muller Arteriosclerosis, Thrombosis, and Vascular Biology, 2011, 10.1161 ATVBAHA 111.237784


Cell Culture Surfaces . . . . . . . . . . . . . . . . . . . 172 Cell Culture Geometry . . . . . . . . . . . . . . . . . . . 176 Compatibility of Surfaces with Microscopy Techniques . . . . . . . . . . . . . 178

Technical Automated Aspects Image of Microscopy Analysis

Optical Properties for High Resolution Microscopy . . . . . . . . . . . . . . 170


Optical Properties for High Resolution Microscopy Focus inside the substrate

Focus above the coverslip

1 mm

~170 µm

x100

x100

Light

Light

CCD-camera

CCD-camera

No. 1.5 Coverslip-Like Thickness The thickness of the coverslip is a crucial aspect of imaging quality. The typical thickness of a coverslip is 0.17 mm (170 μm, No.  1.5). Most of objective lenses used for microscopy are corrected to this special thickness. Thinner or thicker substrates require the use of correction collars on the objective lenses, which then prevents the formation of blurred images by spherical and chromatic aberrations. The special plastic material (ibidi Standard Bottom) that is used in all ibidi µ-Slides, µ-Dishes, and µ-Plates provides coverslip-like optics for high resolution microscopy. The µ-Dishes 35 mm, high, the µ-Slides 2 well | 4 well | 8 well, and 2 well Ph+ | 4 well Ph+ are also available with a coverslip glass bottom (see page 68).

ibidi FREE SAMPLES Order a free sample at www.ibidi.com to test ibidi’s µ-Slides, µ-Dishes and µ-Plates with your experiments.

Compatibility to Immersion Oil

Technical Aspects of Microscopy

θ

170

Immersion oil or water

Objective

θ

Objective

Material Air Water Glycerol Immersion oil Glass ibidi Standard Bottom

Refractive Index (nD 589 nm) 1.0003 1.33 1.47 1.52 1.52 1.52

Identical Magnification – Different Optical Resolution

Low NA Lens

High NA Lens

Objective: 10 x NA: 0.25

Objective: 10 x NA: 0.45

Oil immersion is an important technique that is used to increase the resolution of the objective, up to its physical limit. Placing immersion oil instead of air between the objective lens and the coverslip allows a significantly higher amount of light to be collected by the lens. This, in turn, increases the resolution and the signal-to-noise ratio in microscopic images. Cell culture chambers for high resolution microscopy need to be compatible with immersion oil. All ibidi µ-Slides, µ-Dishes and µ-Plates fulfill this important requirement.

Standard Refractive Index The refractive index n of a material measures the speed of light inside that specific material, as compared to the absolute vacuum. The refractive index is often referred to as “optical density”. For optimal microscope images, the refractive index needs to be 1.52. Like glass and immersion oil, the ibidi Standard Bottom has a refractive index of 1.52.

Numerical Aperture The numerical aperture (NA) of an objective lens summarizes the range of angles over which a lens can collect light. NA is one of the main parameters of an objective lens, which defines resolution and luminous intensity. The numerical aperture is defined by NA = n sin θ, where n is the refractive index of the medium in which the lens works (e.g., 1.52 for immersion oil) and θ is the half-angle of the maximum cone of light that can enter into the lens.


Material Dispersion / Abbe Number > 55

Material

Thickness

ibidi_standard ibidi_UV_special glass_D263 polystyrene quartz

60 40 20 0 200

300

400

500

600

700

visible light

UV C UV B UV A

800 IR

Wavelength [nm]

1.000

Background fluorescence of different materials for microscope slides

900 800 700 600 500

360 nm

400

480 nm

540 nm

300 200 100

rz la ss s id pe is c ta ia Po nd l lys ard ty re ne

ua

G

V

Q

ib

iU

ib

ib

id

rz la s id spe s is c ta ia Po nd l lys ard ty re ne

ua

G

V

Q

iU

id

ib

id

iU

Q

ua rz G l ib V s ass id pe is c ta ia Po nd l lys ard ty re ne

0

ib

Autofluorescence is a material property that describes the intrinsic fluorescence value of the pure material. The fluorescence signal comes from the material and contributes to the imaging process as noise. This can be annoying when trying to image faint fluorescence signals. Depending on the wavelength and type of material, all materials show some degree of autofluorescence signals.

80

Autofluorescence Influences the Signal-to-Noise Ratio

ibidi Standard Bottom: Low autofluorescence Refractive Index nD (589 nm)

Culture Flask: High autofluorescence

Autofluorescence

Abbe Number

ibidi Standard Bottom

# 1.5

1.52

Low

56

ibidi UV bottom

# 1.0

1.53

Very low

58

Glass coverslip D 263 M

# 1.5

1.52

Low

55

Quartz glass coverslip

# 1.5

1.45

Very low

67

Polystyrene (normal Petri Dishes and culture flasks)

Various

1.56

High

33

PC

Various

1.59

High

31

#1.5

1.49

Medium

57

PMMA

Optical Properties for HR Microscopy

Low Fluorescence Background (Autofluorescence)

Transmission [%]

Transmission is a crucial parameter for microscopy. It describes a material’s ability to permit the passage of light through it at specific wavelengths. The more light that is absorbed, the less it can contribute to fluorescence excitation and image acquisition. Unlike normal cell cultureware, which is not transparent in UV light region below 300 nm, the ibidi Standard Bottom also permits light transmission in wavelengths below 300 nm.

100

Intensity in thousands [CPS]

Transmission

Live Cell Imaging

Material dispersion is defined as a variation in the refractive index, depending on the wavelength. In other words, dispersion is a measurement for chromatic aberrations. The Abbe number summarizes the dispersion into one value. It is calculated from the refractive indices of three different wavelengths. The higher the Abbe number, the better the optical quality for microscopy. A material with an Abbe number larger than 55 is considered to be well suited for high resolution microscopy. The ibidi Standard Bottom has an Abbe number of 56 in comparison to D 263M Schott borosilicate glass, which has an Abbe number of 55.

171


Cell Culture Surfaces The successful growth, development, and signaling of cultured cells strongly depends on the surface upon which the cells have been seeded. ibidi μ-Slides and μ-Dishes are perfectly suited for cell-based assays, used in combination with high-resolution microscopy, and can be treated similarly to standard plastic labware, without compromising the imaging quality.

ibidi Standard Bottom The ibidi Standard Bottom, which is our typical surface, is a hydrophilic, foil-like coverslip that forms the bottom of most ibidi μ-Slides, μ-Dishes, and μ-Plates. The Standard Bottom meets all of the optical requirements for various imaging techniques, such as phase contrast, confocal fluorescence, or two-photon microscopy.

Technical Aspects of Microscopy

Human primary keratinocytes on ibiTreat

172

ibiTreat – Tissue Culture Treated – ibidi’s Most Recommended Surface ibiTreat is a physical surface modification that makes surfaces hydrophilic and adhesive to virtually all cell types. The surface is comparable to standard cell culture flasks and Petri dishes. It has been tested with a vast range of different cell lines and primary cells, and is used in over 90% of the more than 2000 peer-reviewed publications that cite the use of ibidi products. Most cells grow well on ibiTreat without the need for any additional coating. Hydrophobic, Uncoated Our uncoated surface does not permit direct cell growth. Due to its hydrophobicity, it can be used for specific coating procedures, or for non-adherent cells. This bottom material is a hydrophobic version of the ibidi Standard Bottom and has identical optical properties.

Normal Petri dish

ibidi Standard Bottom Surface roughness measurement by AFM (atomic force microscopy).

Glass Bottom ibidi offers specialized products with a borosilicate coverglass bottom. They use a standard coverslip glass, which is D263M Schott glass. Glass bottoms are suitable for direct cell culturing, but may require a surface coating to promote cell attachment. Glass can also be coated prior to cell seeding when using adherent cells. For the highest imaging quality, only coverslips with a thickness range of 170 µm +/- 5 µm are used. This thickness, also known as No. 1.5H (high performance), provides the highest precision available that is necessary for modern, high-performance microscope objectives. ibidi developed these glass surfaces especially for TIRF, single photon, and single molecule applications. Examples are the µ-Dish 35 mm, high glass bottom (see page 62) and the µ-Slide 2 well | 4 well | 8 well glass bottom (see page 68).


Coatings

ibidi APPLICATION NOTE

ibidi offers precoated surfaces on the ibidi Standard Bottom. Please contact us for more coating options.

In our Application Note

Collagen IV

Cell Culture Coating (AN 08) you will find detailed information on how to do your own coating on µ-Slides.

Poly-L-Lysine (PLL) / Poly-D-Lysine (PDL) PLL / PDL are polymers of the amino acid L / D-Lysine. This polymer is one of the most commonly used adhesion substrates. It is suitable for a large variety of cell types, especially for neuronal cultures. Adhesion, using this polymer, is mediated by an integrin-independent mechanism. PDL-coated μ-Slides are only available upon request. Delivery time is estimated at three weeks. Please contact us for further information.

RAT1 on Collagen IV

Live Cell Imaging

Collagen type IV is the type of collagen found primarily in the basal lamina. Collagen IV substrates have been tested for a variety of standard cell lines, such as epithelial, endothelial, nerve, and muscle cells. ibidi μ-Slides are coated with a mouse Collagen IV.

Fibronectin Fibronectin is a glycoprotein that is widely used in cell culture coatings. It plays an important role in cell surface interactions that are mediated by the RGD motive. Fibronectin allows neurite outgrowth, and has been used for glial and neural cells. ibidi μ-Slides are coated with a human Fibronectin. MDCK cells on Fibronectin

ibidi Standard Bottom vs. ibidi Glass Bottom ibidi Standard Bottom

Optical properties Refractive index (nD 589 nm) Abbe number Thickness Material Autofluorescence

ibiTreat

Uncoated 1.52

ibidi Glass Bottom

Glass 1.52

56

55

#1.5 (180 µm)

#1.5H (170 µm)

Microscopy plastic

D 263M Schott borosilicate glass

Low

Low

Very high (even ultraviolet)

High (ultraviolet restrictions)

Low (DIC compatible*)

Low (DIC compatible*)

ibiTreat – tissue culture treated Uncoated – hydrophobic

Only pure glass

Protein coatings

Possible

Possible

Gas permeable

Yes

No

Material flexibility

High

Low

No

Yes

All kinds of fluorescence microscopy

TIRF and single photon

Transmission Birefringence (DIC)

Cell Culture Surfaces

Due to its short shelf life, Fibronectin coated μ-Slides are only available upon request. Delivery time is estimated at three weeks. Please contact us for further information.

Other aspects Surface modifications

Breakable Recommended for

* Special glass DIC lids for µ-Dishes and open µ-Slides are available separately (see pages 62 and 68).

173


Cell Culture Surfaces Elastically Supported Surface (ESS) Surface Elasticities in Tissue

174

Stiff

H

ss la

ib

ar

Pe t

d

ri

G

di

sh es id iS Bo ta tto nd m ard

ue ss

ss

m iu

ed

M

So

ft T

Ti

is

su

e

ue

Soft

Ti

GPa MPa kPa

1012 11 10 10 10 9 10 8 10 7 10 106 105 104 103 102

1.5

ue Ti ss

cl us M

nn

ec

tiv e

G

e, E En pit do he th lia, el ia Ca rti la Bo ge, ne

1

a la in, nd

28 15

Co

Please note that the morphology of the cells might look different on the ESS surface, when compared to the standard culture on a stiff surface.

10

Br

NOTE

100

ESS Products [kPa]

The tissue elasticity differs greatly within the mammalian body. There are very soft tissues, such as neuronal cells, brain, or gland tissue that exhibit elasticities between 1 and 5 kPa. Then, there are the hard tissues, such as cartilage or bone, with values up to 100 kPa. However, most body tissues show in-between values in the range of 10-30 kPa.

Surface elasticity [kPa]

Technical Aspects of Microscopy

Tenocytes grown on a µ-Dish 35 mm high ESS, 15 kPa (see page 63).

Surface elasticity [Pa]

Cells on ESS can interact with the surface and deform it.

In tissue, cells grow under different conditions, such as those commonly found in in vitro cell cultures. A crucial parameter that effects cell proliferation, differentiation, and overall function, is the surface stiffness/elasticity. Recent research has shown significant evidence that this physical parameter must be taken into consideration. The elasticity (Young’s Modulus) of most plastics used for cell culture is around 1 GPa. Young’s Modulus of glass is approximately 70 GPa. In contrast to plastic and glass, the Young’s Modulus of mammalian cells is between 1 and 100 kPa which means that the natural cell environment is at least 100,000 times more elastic.

ibidi’s elastically supported surfaces ESS (as used in the µ-Dish 35 mm, high ESS, see page 63) are available in three different elasticities, 1.5 kPa, 15 kPa, and 28 kPa. These three different elasticities cover the whole range of elasticities that is found in tissue in vivo, from soft tissue (brain, gland) to hard tissue (cartilage, bone).


ibidi µ-Dishes ESS

Cell medium

The ESS Dishes are based on ibidi’s popular µ-Dish . A 40-µm, highly elastic material (biocompatible silicone) is coated onto a 100 µm-thin glass cover slip. The whole bottom provides a thickness of 140 µm and a very high optical quality. Using the µ-Dishes ESS (Elastically Supported Surface), it is possible to combine high-end Microscopy (Confocal, High Resolution, Fluorescence, etc.) with a close-to-nature, 2D soft surface environment. The uncoated ESS surface is very hydrophobic and does not allow for any direct cell growth. For an easy cell culture, we recommend using specific, extracellular matrix coatings, such as Collagen or Fibronectin. 35 mm

Elastic surface

0.04 mm

Glass support

0.1 mm

100 x

In ibidi’s µ-Dish 35mm, high ESS cells grow on a soft and elastic 2D surface, which mimics the elasticity of tissue. High resolution microscopy is possible, due to the high optical quality of the µ-Slide (low autofluorescence, thickness of 140 µm).

Live Cell Imaging

Cells attached to our ESS surface can interact with that surface and deform it. This leads to a more in vivo-like cell behavior, especially for cell types related to mechanical forces, like muscle cells. Recent publications have also shown that the surface elasticity has a tremendous influence on cell differentiation and migration.

Coating

Undifferentiated Fibroblasts on Glass Surface (70 GPa)

Cell Culture Surfaces

Zyxin

a-smooth muscle actin

Differentiated Fibroblasts on Elastic Surface (28 kPa)

ibidi FREE SAMPLES

Zyxin

a-smooth muscle actin

Order a free sample at www.ibidi.com to test ibidi’s µ-Dishes ESS with your experiments.

175


Cell Culture Geometry

Open Well Format

Cell Culture Channel

Refined Geometry

• Common formats

• Excellent imaging

• Easy handling

• Easy liquid exchange

• Large volume

• Low volume

• Designed for special purposes (e.g., gradients or gel matrices)

All-in-One Chambers All-in-one chambers reduce the time and number of experimental steps in cell-based assays. ibidi µ-Slides, µ-Dishes and µ-Plates are designed as all-in-one chambers, so that all of the experimental steps, from cell cultivation to microscopic imaging, can be done in one chamber.

Technical Aspects of Microscopy

µ-Slide 8 well (see page 66)

µ-Dish 35 mm, high (see page 61)

The open well format of the ibidi µ-Slide 2 well | 4 well | 8 well (see page 66) and the µ-Dish 35 mm, high (see page 61) allows for the use of standard immunofluorescence protocols. After staining the sample can be observed through the coverslip-like bottom, using high resolution microscopy. There is no need for an additional coverslip. Channel formats like the µ-Slide VI 0.4 (see page 78) are ideal for an exact exchange of small amounts of reagents. These µ-Slides reduce the number of experimental steps needed in immunofluorescence assays. The channel format supports homogeneity of cell distribution and phase contrast microscopy.

ibidi µ-Slides, µ-Dishes, and µ-Plates are Designed as All-in-One Chambers. µ-Slide VI 0.4 (see page 78)

ibidi FREE SAMPLES

1) Cultivation

2) Fixation

3) Staining

4) Imaging

Order a free sample at www.ibidi.com to test ibidi’s µ-Slides, µ-Dishes and µ-Plates with your experiments.

176

100 x


Compatibility of ibidi Products with Upright or Inverted Microscopes Upright Microscope

Inverted Microscope

100x

• 12 well Chamber, removable (see page 85)

• All other ibidi μ-Slides, μ-Dishes, and μ-Plates

• μ-Slide VI – flat (see page 80)

• ibidi Heating System (see page 15)

In cell biology, upright microscopes are used for:

Inverted microscopes are popular for live cell imaging, because:

• Samples squeezed between a slide and coverslip • Fixed samples, such as cells and tissue sections Not recommended for live cell imaging.

• Cells sink to the bottom and onto the coverslip for adherence

Live Cell Imaging

100x

• Sample access from the top, e.g., for liquid exchange or micropipettes

Homogeneous Cell Distribution, as a Result of Geometry

Inhomogeneous cell distribution in open wells

Homogeneous cell distribution in cell culture channels

Phase Contrast in Channel Slides is Superior to Small Open Wells In phase contrast, small phase delays that come from slightly different refractive indices in different materials are converted into intensity changes over the entire image. Phase contrast microscopy in channel slides (e.g., ibidi’s µ-Slide VI 0.4, see page 78) is much better than in small open wells.

96 well plate or small open well: Strong influence of meniscus, low contrast near the edges

Channel or parallel plates: No meniscus, good phase contrast over the entire area

Cell Culture Geometry

Cell densities in open wells are very dependent on handling during cell seeding. Unlike in the open wells, cell densities in channels neither vary with the position inside the slide, nor with the handling and treatment during and after cell seeding.

177


Compatibility of Surfaces with Microscopy Techniques Phase Contrast Phase contrast is a technique that converts small phase shifts in cells into amplitude or intensity contrast. This label-free technique is strongly dependent on having the right alignment of the phase plate and the annular ring in the optical pathway. It is by far the most frequently used method in biological light microscopy. A common problem in phase contrast is the meniscus formation at the air-liquid interfaces, thus making this technique nearly non-applicable to small culture wells, like 96 well plates.

ibidi’s recommendation: Phase contrast is rarely dependent on the material. What is crucial is the meniscus, which is formed in small open wells. Therefore, channel µ-Slides or µ-Slides Ph+ are great tools for phase contrast.

DIC – Differential Interference Contrast DIC is a label-free microscopy technique with a high sensitivity to thin cellular material, even when it is located within thick tissue. It is less sensitive to meniscus formation than phase contrast. DIC needs low birefringence and is, therefore, not compatible with standard culture ware made out of polystyrene.

ibidi’s recommendation: DIC is 100 % compatible with the ibidi Standard Bottom and Glass Bottom. There are drawbacks in using DIC with regular channel slides and plastic lids. Therefore, ibidi offers special DIC lids for µ-Dishes (see page 62) and µ-Slides (see page 68).

Technical Aspects of Microscopy

Wide-Field Fluorescence

178

Wide-field fluorescence microscopy is a form of light microscopy. The specimen is illuminated with filtered light at wavelengths that excite fluorophores. It requires labeling that uses special antibody reactions, or tagged proteins (e.g., green fluorescent protein). Fluorescence is used to detect structures, molecules, or proteins within the cell, up to highest magnifications. ibidi’s recommendation: The ibidi Standard Bottom is optimized for fluorescence microscopy, making wide-field fluorescence possible without restrictions.

 Confocal Microscopy Confocal microscopy is based on conventional wide-field microscopy. Laser light is focused into the sample, exciting only a small spatial area. Pinholes inside the optical pathway cut off signals that are out of focus, thus creating images of one, single optical plane. With this technique, it is possible to create 3D images from data that was generated from several optical planes.

ibidi’s recommendation: The ibidi Standard Bottom is optimized for fluorescence microscopy, so that confocal microscopy is possible without restrictions.

TIRF – Total Internal Reflection Fluorescence TIRF utilizes the evanescent field, which is created when a beam of light strikes an interface between two media, exciting the fluorescent dyes in the specimen. Although TIRF cannot image deep into a specimen, it allows imaging of the specimen near the interface with a high signal-to-noise ratio. This technique requires two optical media with different refractive indices, such as glass (n=1.52) and water (n=1.33).

ibidi’s recommendation: TIRF is possible with the ibidi Standard Bottom, but we recommend using a glass bottom.


ibidi Standard Bottom

ibidi Glass Bottom

Standard Polystyrene plates & dishes

FRET – Förster Resonance Energy Transfer

ibidi’s recommendation: The ibidi Standard Bottom is optimized for fluorescence microscopy, making FRET microscopy possible without restrictions.

FRAP – Fluorescence Recovery After Photobleaching FRAP is a fluorescence microscopy method to study the mobility of fluorescently-labeled molecules. A typical FRAP experiment involves three distinct phases. After the registration of the initial fluorescence, the fluorescent molecules are photobleached within a selected area using the laser beam. Next, the fluorescence recovery is recorded when it arises from the immediate surroundings, by diffusional or active transport, from the exchange between photobleached molecules and intact ones. Then it is possible to obtain the diffusion coefficient and a local (im)mobile fraction, using modeling. ibidi’s recommendation: The ibidi Standard Bottom is optimized for fluorescence microscopy, making FRAP possible without restrictions.

In contrast to normal fluorescence microscopy, where the intensity is used to create an image of the specimen, FLIM uses the lifetime of the signal by analyzing the fluorophore’s exponential decay rate. By detecting differences in lifetime, it is possible to identify fluorophores that have the same excitation and emission spectrum. The fluorescence lifetime is dependent on ion intensity, oxygen concentration, molecular binding, and molecular interaction. However, FLIM signals are independent of dye concentration, excitation light intensity, and photobleaching. ibidi’s recommendation: The ibidi Standard Bottom is optimized for fluorescence microscopy, making FLIM microscopy possible without restrictions.

2-Photon Microscopy 2-Photon microscopy is based on the fact that a fluorophore can be excited when it is hit by two photons at the same time (typically within several femtoseconds). Typically, the wavelength of the two photons doubles the normal excitation wavelength, so that the excitation energy values are combined, causing a fluorescence signal. The probability of finding two photons, at the same time and at the same spot, is only likely in the focal plane of high numerical aperture objective lenses. The high excitation wavelength is less phototoxic, and it enhances the penetration depth when imaging a thick tissue material. ibidi’s recommendation: The ibidi Standard Bottom is optimized for fluorescence microscopy, making 2-Photon microscopy possible without restrictions.

Super-Resolution New optical microscopy methods such as STED, SIM, (F)PALM and (d)STORM bypass the diffraction barrier and enable super-resolution imaging or “Nanoscopy” with substantially improved optical resolution. Nowadays they can provide a spatial resolution in three dimensions that is well below the diffraction limit and close to a near-molecular resolution. They can be applied to biological samples, and provide new views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales. ibidi’s recommendation: Super-Resolution is possible with the ibidi Standard Bottom, but we recommend using a glass bottom. (Example provided by Markus Sauer, Würzburg)

Compatibility with MicroscopyTechniques

FLIM – Fluorescence Lifetime Imaging Microscopy

Live Cell Imaging

FRET is a fluorescence technique that determines the precise location and nature of the interactions between fluorophores within living cells. A donor fluorophore in its excited state can transfer its excitation energy to an acceptor fluorophore in a non-radiative fashion. Typically, this happens through dipoledipole coupling in a distance of less than 10 nm. Beyond that distance (Förster radius), the two fluorophores show normal fluorescence behavior.

179


180


ibidi Application Notes . . . . . . . . . . . . . . . . . . 182 ibidi Movies . . . . . . . . . . . . . . . . . . . . . . . . . . 183 ibidi Practical Course: Chemotaxis Assays and Video Microscopy . 184

ibidi Webinars . . . . . . . . . . . . . . . . . . . . . . . . 186 ibidi Reference Database . . . . . . . . . . . . . . . . 187 Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . 188 Catalog Number Index . . . . . . . . . . . . . . . . . . 190

ibidi Support

ibidi Practical Course: Cell Cultivation Under Perfusion and Live Cell Imaging . . . . . . . . . . . . . . . . . . . . . . 185

181


www.

.com

ibidi Application Notes During studies run solely and in cooperation with scientific partners, ibidi has produced numerous application notes. Together with over 3000 scientific papers, these application notes form part of a powerful body of work that is available for your research purposes from our website www.ibidi.com.

Application

Application

Note 08

Coating procedu

res for ibidi

Note 08

Use the following

μ-Slides

protein concentra

Channel Slides and μ-Dishe s For optimized cell Collagen IV μ-Slide I family. The ibiTreat adhesion there are different 75 treatments and μ-Slide I 0.1 surface is comparab This surface coatings Luer le with standard permits direct the μ-Slide300 μ-Slide I 0.2for tissue culture cell growth primary cells. treatedLuer as shown with Compared to plastic ware.150 a large μ-Slide I 0.4 Luer ibiTreat, uncoated coated with adhesion 75 0.6 cell lines μ-Slide I of has a hydrophob number factors for the Luer and ic μ-Slide surface,0.8which adhesion of most 60 I Luer must be cells. 1. Recomm 38 μ-Slide III 0.1 ended surfaces 300 μ-Slide III 3in1 For Collagen 75 IV: hydrophob μ-Slide VI 0.4 ic, uncoated 75 μ-Slide VI 0.1 For Fibronecti 300 μ-Slide VI flat n: hydrophob ic, uncoated 75 μ-Slide y-shaped For Poly-L-Lys 75 μ-Slide V ine: ibiTreat (tissue 75 culture treated) μ-Slide upright 0.7 60 For Poly-D-Ly μ-Slide Chemotaxi 1) sine: ibiTreat s 45 (tissue culture μ-Slide Chemotaxi 2) treated) s 100 μ-Slide Chemotaxi If you want 1) s to do a different μ-Slide Chemotaxi 3D 40 ibiTreat and uncoated. coating by yourself, s 3D2) 70 Some products we recomme nd trying both are also offered surfaces, Open Formats with glass bottom. Please note that there is no ibiTreat version μ-Plate 384 well. Collagen IV of μ-Slide V, In those cases, μ-Slide I 0.1 Luer, use the hydrophob μ-Slide μ-Dish 35mm, low ic, uncoated III 0.1, and surface 15 35mm, μ-Dishfor 2. Prepare the allhigh 3) coatings. coating solution

tions [μg/ml]: Fibronectin 75 300 150 75 60 38 300 75 75 300 75 75 75 60 45 100 40 70

Fibronectin

20 20 25 15 17 17 50 15 30 15 25 47

15 All coating solutions 18 μ-Slide 8 well 18 are calculated recommended 11 μ-Slide 2x9 well for a certain by the manufactu 11 amount of protein 12 rer’s reference μ-Slide per 18 well 12 . area 12 μ-Slide Angiogene (μg/cm²) For Collagen 12 sis IV: (1.5 38 2 μ-Plate 96 well Dilute the Collagen μg/cm ) 38 12 μ-Plate 384 well IV (e.g. Becton-Di 12 concentration ckinson, mouse 24 using 0.05 M r 12 well tumor, No.μ-Chambe 24 HCl. 11 356233) to the Culture-Ins ert 11 desired For Fibronecti 18 Culture-Ins n: (1.5 μg/cm2 ert StemCell 18 ) Dilute the Fibronecti 35 35 n (e.g. Becton-Di 1) concentration ckinson, human when coating using PBS (pH full chamber plasma, 2)354008) 7.2) without 2+ when coating Ca and Mg2+ to theobservatio desired n area . 3) For Poly-L-Lys also valid for only ine: 2 glass bottom Dilute the PLL (2 μg/cm ) and ESS versions (e.g Sigma-Ald concentration rich. 0.01% using ultra pure solution, 100 μg/ml, P4832) water. to the desired For Poly-D-Ly sine: (5 μg/cm2 ) Dilute the PDL (e.g. Becton-Di ckinson, No. pure water. 35 4210) to the desired concentra tion using ultra

Application Note 08 © ibidi GmbH, Version

3.4, Elias Horn,

Application Note 08 © ibidi GmbH, Version

May 17th, 2010

3.4, Elias Horn,

Poly-D-Lysine 250 1000 500 250 200 125 1000 250 250 1000 250 250 250 200 150 330 130 230

Poly-L-Lysine

15

15

μ-Dish 50mm, low

Poly-L-Lysine 100 400 200 100 80 50 400 100 100 400 100 100 100 80 60 133 55 90

Poly-D-Lysine 50 50 60 35 40 40 125 35 70 35 60 115

May 17th, 2010

Page 1 of 4

Page 2 of 4

Cell Culture Coating (AN 08) Provides detailed information on how to apply your own coatings onto µ-Slides.

Application

Application

Note 09

Note 09

Application

2) Fixation of cells  Aspirate medium Wash cells with from all reservoirs using a cell culture Dulbecco´s PBS aspiration device. reservoir of each by slowly applying channel and 200 μl into one channel. Don’t aspirating from with μ-Slide empty aspirate the entire the opposite VI 0.4  Fix cells with reservoir for In this protocol channel volume. each ~100 μl of 3.7 we describe % para-form the liquid inside inside the μ-Slide a single example -aldehyde in the channel by VI 0.4 PBS. After Fluor® 488 phalloidin . Subsequently, we stainedof cultivating HT-1080 the filling one well content of the cancer with 200 μl PBS 10 min flush reservoir from the F-actin cytoskele dry. cells and counterst the other well; and removing ained the nucleus ton with Alexa ensuring the with DAPI. channel is never The protocol consists of four Permeabilization main steps: and blocking  Wash cells again with 200 μl PBS as described  Apply ~100 μl of 0.1% Triton® above.  Wash cells X-100 (Fluka) in PBS with PBS. for 3-5 min.  Apply ~100 μl of 1% BSA in PBS solution  Wash cells for 20 min. with PBS. 3) Staining  Remove all liquid from the channel let the channel using your dry. aspiration device. Don´t  Right after that, apply 25 μl of Alexa Fluor® PBS + 1% BSA, 488 phalloidin Invitrogen Corp.) (1 Unit + 500 Incubate at room μl temperature  Wash cells for 20 min. with PBS.  Apply 25 μl DAPI (0.1 μg/ml, Sigma-Ald n rich) for 3-5 min.  Wash cells Unpack a μ-Slide with PBS and VI 0.4, ibiTreat apply filled (ca. 50 it on a μ-Slide (80606) under μl). ibidi Mountingibidi Mounting Medium rack (80003). sterile conditions until the channel DABCO Apply Medium is suspension and for put anti-fading*. The is glycerol based into each channel. 30 μl of a 3x105 cells/ml slide can be and contains HT-1080 cell illustrated below Pipet directly stored for approx. 4) Imaging or shown on into the 4 weeks. channel as our website.  Observe cells under a fluorescence and optionally microscope with immersio with appropriat n oil. e filter sets

  

Tables on page

[

13

dyn ]  2.274 Φ[ ml ] cm2 min

γ[s ]  227.4 ml Φ[ ] min 1

[

dyn ]  1.137 Φ[ ml ] cm2 min

γ[s1]  113.7 ml Φ[ ] min

[

dyn ]  7.741 Φ[ ml ] cm2 min

γ[s1]  774.1 ml Φ[ ] min

dyn ]  2.274 Φ[ ml ] cm2 min

Shear stress

[

Shear rate

γ[s1]  227.4 ml Φ[ ] min

____________ ____________ ____________ ____________ Application Note Application Note ____________ ____________ ____________ ____________ 11 11 ____________ ____________ © ibidi GmbH, © ibidi GmbH, ____________ ____________ Version 3.4, Version 3.4, Roman Zantl, Roman Zantl, ____________ ____________ October 27th October 27th ____________ ____________ 2010 2010 _____ _____

Page 2 of 2

Page 3 of 18

Page 1 of 18

Fluorescence Staining (AN 02 / 09 / 15)

Shear Stress and Shear Rates (AN 11)

These protocols provide instructions on immunofluorescence staining using various µ-Slides.

Provides detailed information on shear stress / shear rates and flow rates in ibidi’s channel slides.

Application

Application

Note 13

Flow experim

ent with the

Application

Note 13

6. Connecting

ibidi pump system

the Slide with

the perfusion

and

Note 13

© ibidi GmbH,

____________

Version 3.0,

Prepared by

____________

Helga Wagner,

Note 13

____________

March 18th,

© ibidi GmbH,

____________

____________

Version 3.0,

____________

2011

____

Prepared by

Page 1 of 8

____________

Helga Wagner,

____________

March 18th,

____________

2011

Application

Note 14

2D Chemotaxis set:

Place the fluidic µ-Slide I 0.6 Luer unit with the 1. General informati the sterile work mounted perfusion on bench and pinch The following set under using the plastic application note off the tubes near the valve clip (a). Put the combining the describes the surface, take µ-Slide I Luer ibidi pump system performance off the caps recommendations of a perfusion until with a µ-Slide and fill the reservoirson the working 0.6 there is a small assay for the usage Luer. Furthermo with medium are given. This Pull out the first of human umbilical I hump of liquid re somemale Luer protocol can (see the small vein endothelia be adapted for adapter from tol make cells (HUVECs) sure there are the middle connectorpictures). your special no air bubbles experimental For the setup ondemands. holding it upwards the slide tipping remaining inside. you need the it cautiously (b) Connect it to following material: as you see in • µ-Slide I 0.6 the female Luer the figures (c Luer, ibiTreat - f). • ibidi pump system - air pressure pump - fluidic unit - perfusion set, 15 cm, ID 1.6 mm (red marking) - hose clip • HUVECs • Endothelia l Cell Growth Medium 2. Cell Culture Cultivate your cells according to your normal Endothelial Cell protocol. Growth Medium 10% fetal calf (PromoCell, Germany, For HUVECs we recomme serum (FCS). nd C-22010) suppleme confluence when Always take nted with care that the cells are just period agglomera starting a new experimen t. Cells that reaching te Repeat this are confluent procedure with the cells homogen very firmly and you might the second male a longer encounter difficulties withover ously. Another a wipe (see figure g Depleted cells Luer adapter. important aspect by suspendin - l). may not endure Then remove g is the the overspill not use HUVEC shear stress and will be flushed fitness of the cells. at more than passage 4 (split from the surface. ratio 1:2)! Do 3. Preparation of the material Equilibrate all needed material overnight inside like slides, medium the incubator and tubing (perfusion bubbles emerging at 37°C and 5% CO . This sets) over time. 2 is essential to avoid air Place the perfusion set on the fluidic instructions and unit as described fill in about 12 in the both reservoirs ml of equilibrate should be equilibrate d medium (withoutibidi pump system get out the air d at 5 ml after slide). The level bubbles from removing all the tubes start predefined setup air from the tubes. of a medium flow can be loaded To cycle. For this “Tutorial” and in the Pump purpose a choose “Load Control software. demo setups” Go to the menu Æ “Remove point Pressure air bubbles”. Shear stress 1) 50.0 mbar Flow rate Take care you None (no slide) While running Time span work as fast 23.4 ml/min the program, and careful as any disturbanc gently flip on the air bubbles. possible. The infinite e and will detach the tubes and cells are stressed if there is too on the adapters Check at much agitation. your cells on to remove the microscop It is crucial, that e after connectio all bubbles are n to the tubing! Gas remaining gone before adapting the in the system slide to the fluidic can influence stop the flow. the flow rate unit! and in the ____________ ____________ worst ____________ ____________ case Application Application ____________ ____________

ibidi Support

γ[s1]  176.1 ml Φ[ ] min

(3 mm channel)

H., Kim M., Kim M., Kim J. & Chung Y., Lee S.Y., Karess R.E., Nature Aug 2007; J.; Energy-dependent regulation Lee S.-H., Shong doi:10.1038/nature0582 of cell structure M., Kim J.-

assay using

   

Note 14

4. Coating μ-Slide

μ-Slide Chemot

0. Importan t Notes

axis

Read the related document “Important Follow all steps Notes” first. in this protocol Watch the handling carefully. movie on www.ibidi Start with free .com. samples and the included food coloring.

1. General Informati

on The μ-Slide Chemotaxis is a tool for chemotactical observing responses of adherent migrating in 2D over extended periods cells concentration of time. The profile which linear chemotactical is required movement is for generated by and stable for diffusion at least 48 hours. one slide allow Three chambers parallel chemotaxi on following protocol s experimen ts. The is adapted for adherent cells only. Color code for liquids in this Application Note:

Chemotaxis

μ-Slide Chemotaxi (80301) permits s with hydrophobic, uncoated no direct cell surface to be coated. growth. The surface needs For direct cell treated version growth the tissue μ-Slide Chemotax precoated μ-Slide is, ibiTreat (80306)culture Chemotaxis, or the can be used. Collagen IV, Work under sterile sterile can be adapted conditions. Other (80302) by using the coatings same procedure. a) Full Coating Protocol with Collagen IV Please use the following protocol of the chamber to coat the entire with inside (uncoated μ-Slide, Collagen IV. 0.8 μg/cm2, 80 μl, 24 μg/ml, 2.4 cm2):  Dilute the Collagen IV (e.g. BD Cat.-Nr. 356233) a concentration of 24 μg/ml using to 0.05 M HCl.  Inject 80 μl coating solution per chamber filling port using 5 (step A) until the chamber the completely filled. Take care to is filling ports. fill all channels and 

Leave at room Chamber configurati temperature for at least 30 on:the caps Use 1 Observation minutes. to decrease evaporatio area 2 Large reservoir (2000x1000 μm2) n (step B). (40 μl) 3 Large reservoir Completel y aspirate (40 μl) the solution 4 Filling port culture aspirator. using a cell with small Take care to side channel 5 Filling port than aspirate from one filling port with small more side to remove as 6 Filling port possiblechannel much liquid (step C). as 7 Filling port 8 Plugs  Wash with 80 μl ultra-pure 9 Cultivation caps water by injecting pipet ports 5 and 7 (step D). into Please note the chamber that

with water filling or washing difficult as long 2. Principle buffer is as mind air bubbles it is not completely dried. Two large volume Don’t at this point. different chemoattr reservoirs are connected by a thin slit. actant concentrat  Aspirate connecting slit The reservoirs exhaustively ions (indicated a linear and stable (2, by using a 3) contain below by red aspirator. cell culture concentration Take care to aspirate from profile is generatedand blue color). Inside filling more than one portthe to remove as by diffusion. much liquid (step E) as possible. 

Repeat until the entire inside is washed. Let the chamber dry at room overnight under temperature sterile conditions .

Cross section indicating the cells, chemoattra place ctant, and gradient of Application Note 14 © ibidi GmbH, Version

2.5, Elias Horn,

September 1st

Please note that filling the chamber washing buffer with cells is difficult as or dried. long as it is not completely Application Note 14 © ibidi GmbH, Version

2011

____________

____

2.5, Elias Horn,

September 1st

2011

Page 1 of 15

Page 4 of 8

Page 3 of 15

HUVECs under Perfusion (AN 13)

Chemotaxis (AN 14 / 17 / 23)

Demonstrates how to set up a flow experiment using the µ-Slide I Luer and Human Umbilical Vein Endothelial Cells (HUVEC).

These application notes provide practical advice on how to run chemotactical assays using the µ-Slide Chemotaxis 2D and the µ-Slide Chemotaxis 3D.







   

Application



                                                                                                                                                                                                                                                                     



           

    

182 182

dyn ]  1.761 Φ[ ml ] cm2 min

[

Shear rate

μ-Slide III 3in1 :

by AMP-activa 8 ____________ ____________ ted protein kinase. ____________ ____________ Application Note Application Note ____________ ____________ ____________ ____________ 09 09 ____________ ____________ Version 1.6, Version 1.6, Elias Horn, January Elias Horn, January ____________ ____________ ____________ ____________ 14th, 2011 14th, 2011 ____________ ____________ _____ _____ Page 1 of 2

:

Shear stress

Shear rate

HT-1080 cell; green: F-actin cytoskeleton; blue: nucleus (Zeiss Axiovert 135; Plan-Neof luar 40x/0.75)

Cover reservoirs with the supplied Put the slides lid. with the rack into the incubator cells attach (60 min). Afterward (37 °C; 5 % CO2) and let free medium. s fill both reservoirs with 60 μl of Incubate over * As published: cellnight. Lee J.H., Koh

Note 11

μ-Slide VI 0.4

Table of content: Formulas for shear stress and shear rates Shear stress – for all calculation with different dynamica ibidi channel slides ............... Ranges of shear ....... 2 stress and flow l viscosity .............μ-Slide y-shape Experimental d: ........................ Aspects................. rates .............................. (single 5 channel area) .............................. Shear Stress .............................. table ........... 6 Shear Rate table for μ-Slide I 0.1 Luer for viscosity ............................................. for μ-Slide I 0.1 ..... 7 =0.01 dyn·s/cm² Shear Shear Stress Luer: ............... stress : ............... table .............................. ...... Shear Rate table for μ-Slide I 0.2 Luer for viscosity ............................ 8 for μ-Slide I 0.2 =0.01 dyn·s/cm² 8 Shear Stress Luer: ............... : ..................... table for μ-Slide 0.4 .............................. Shear I Luer for viscosity Shear Rate table ............................ 9rate for μ-Slide I 0.4 =0.01 dyn·s/cm² 9 Shear Stress Luer: ............... : ................... table .............................. 10 Shear Rate table for μ-Slide I 0.6 Luer for viscosity ............... μ-Slide ........... y-shape for μ-Slide I 0.6 10 d: =0.01 dyn·s/cm² Shear Stress Luer: ............... : ............... table area) .............................. (branched .... 11 Shear Rate table for μ-Slide I 0.8 Luer for viscosity .......................... for μ-Slide I 0.8 11 =0.01 dyn·s/cm² Shear Stress Luer: ............... : ............... table Shear .............................. .... 12 Shear Rate table for μ-Slide VI 0.4 for viscosity .......................... stress =0.01 for μ-Slide VI 0.4 Shear Stress : .............................. dyn·s/cm²: ......................... 12 table 13 .............................. Shear Rate table for μ-Slide VI 0.1 for viscosity Shear ............... =0.01 for μ-Slide VI 0.1 .. 13rate Shear Stress : .............................. dyn·s/cm²: ............... table .......... 14 .............................. Shear Rate table for μ-Slide III 0.1 for viscosity ................. 14 =0.01 for μ-Slide III 0.1 Background : .............................. dyn·s/cm²: ............... information on .......... 15 .............................. the numerical Area of homogen ................. 15 eous shear stress...... calculations ............... Flow profile in μ-Slide ............... .............................. III 3in116 .............. y-direction............... : ...............(1 Shear stress .............................. mm channels) ............... and shear rates ........ 17 .............................. in μ-Slide y-shaped ............... 18 .............................. ....................... Shear 18stress

1) Cultivatio 

Application

Note 11

Shear stress and shear rates for all channel μ-Slides - based on numeric al calculations

Cell cultivati on and immunofluoresc ence staining

  

    

 

  

 

Application

Note 28

Note 28

Protocol for

adenoviral transdu The attachmen t of adenoviru ction of human ses to cells cells Coxsackie-Adenovirus is Receptor (CAR), mediated by high-affini endocytosis 1. General ty binding to upon interaction while internaliza Information ............... the with αv-integrin provided by microtubu tion occurs 2. Backgrou .............................. s. By means through nd .............................. les, the adenoviru .............................. of transport its DNA into 3. Material .............................. s reaches the mechanisms it.... ............... and Equipmen host cell’s nucleus 1 .............................. t Required. 4. Safety and and injects Handling of Recombin ............................................. After ................ 1 5. Experime entering ............... ant Adenoviru nt: Transduct ses ............... integrate ...........the 2 nucleus, the viral DNA ion of HUVECs a. Calculatin into the host remains epichromo with rAVCMV-LifeAct-T ............................. g the amount chromosome 3 host genes). somal (i.e., of virus required agGFP2 b. Preparing and therefore ...............A...simplified in a μ-Slide 8 cells for use does not activate it does not illustration 3 in a μ-Slide 8 well........................ of the infection c. Example or inactivate well ............... results ............... ...... 3 mechanism is .............................. 6. Optional: .............................. shown in Figure Determining ............ and 1. .............................. 3. Material 4 Equipme the MOI........ ............... nt Required .............................. .............................. ...... 4 1. General Informati ........ 5 For this protocol on the following materials are required: This protocol describes the adenoviruses standard technique Name as well as how Concentration for handlingReagent/Material to design an example, a Company recombinant rAVCMV-LifeAct-Tag approach to transduction Order No. transduce human GFP2 experiment Cells (HUVEC) of primary Human 1x1010 IU/ml cells. As an with a recombina μ-Slide ibidi 8 well (ibiTreat) Umbilical Cells shown. nt adenovirus 60121 Endothelia harboring LifeAct-Ta Vein ibidi HUVECs * gGFP2 transgene l 80826 is (*also commercially available) 2. Backgrou Self-prep† Endothelial nd Cell Growth Medium

These days, replication-deficient recombinant adenoviruses (serotype 5) are widely used in research laboratories. This modified adenovirus, where genes E1 and E3 have been depleted, is still able to infect cells. However, the essential genes producing new for viral particles, known as also virions, are no longer present. There are numerous using an adenoviru advantages in s to introduce genetic material into host cells. viruses can These be many mammalia used to transduce n cell types, both (especially human) replicative and replicative. nonMoreover, recombina nt adenoviru can also be used ses to transduce sensitive cells various with low transfectio efficiencies. n Application Note 28 © ibidi GmbH, Version

1.0, Julia Riedl,

September 15th

ECGM supplement Penicillin/Streptomycin

Table 1: Material and

For this protocol     †

the following

reagents needed

equipment and

1x 1x

-

Promocell

C-22010

Promocell

5%

PAA

for the transductio

incl. P11-010

n of HUVECs.

instruments

are required:

Cell culture incubator (high humidity, Tissue culture 37 °C, 5 % CO hood 2) Fluorescence microscope equipped with Emmax 506 nm), a TagGFP2 filter a stage top incubator set (Exmax 483 lapse function (37 °C, 5 % nm / CO2) and optionally Optional, cell , a time culture dishes (e.g., a 24 well plate)

as published

in:

Jaffe, E. A. et al. Culture of Human Endothelial morphologic and immunologi Cells Derived c criteria. Journal from Umbilical Veins – Identificatio of Clinical Investigatio n by n 52, 2745-2756 (1973) Figure 1: Simplified illustration of adenoviral transductio the n mechanism Application.

Note 28 © ibidi GmbH, Version 1.0,

2011

Julia Riedl, September

15th 2011

Page 1 of 6

Page 2 of 6

Tube Formation (AN 05 / 19 / 27)

Adenoviral Transduction of Human Cells (AN 28)

These protocols describe the set up of a tube formation assay, using the µ-Slide Angiogenesis or the µ-Plate Angiogenesis 96 well, and subsequent data analysis.

This is a detailed protocol that explains how to handle recombinant adenoviruses as well as how to design an approach to transduce human cells.


www.

.com

ibidi Movies ibidi movies provide detailed practical information on cell-based assays and cell culture applications. A selection of the available movies has been detailed below. These movies and many more are available from our website www.ibidi.com.

Use of µ-Slides

Seeding Cells in µ-Channels (MV 01)

Exchanging Fluids in µ-Channels (MV 02)

Creating a Concentration Gradient (MV 03)

Performing an Angiogenesis Assay (MV 15)

Tube Formation Assay Evaluated with the Wimasis Automated Image Analysis (MV 23)

Performing a Wound Healing Assay (MV 16)

Wound Healing Assay Evaluated with the Wimasis Automated Image Analysis (MV 22)

Chemotaxis Assays (MV 25)

Immunofluorescence Using the µ-Slide VI (MV 18)

LifeAct: Actin Marker for Live Cell Imaging (MV 19)

Long-Term Cell Culture Under Perfusion (MV 21)

Cell Transfection Using Torpedo DNA (MV 24)

MDCK Cells in a µ-Slide VI 0.4 (MV 13)

Cell-Based Assays

Chemotaxis of HT-1080 in a µ-Slide Chemotaxis 2D (MV 08)

HUVEC Culture Under Perfusion vs. Static Conditions (MV 10)

Application Notes and Movies

Cell Culture Applications

183 183


ibidi Practical Course: Chemotaxis Assays and Video Microscopy 2 day Laboratory Course at ibidi Munich / Germany

Topics: The aim of the course is to learn the experimental setup and analysis of chemotactical assays, by means of video microscopy. The focus is on analysis of chemotaxis of adherent cells in 2D and non-adherent cells in 3D gel matrices. Experiments are performed and analyzed using HT-1080 cancer cells and dendritic cells. Main topics are: sample preparation, video microscopy of migrating cells, cell tracking, data analysis and presentation of the results. Finally, characteristic parameters for the description of directed and / or undirected cell migration are evaluated.

Target Group:

ibidi Support

The course is intended for scientists and technical associates with profound experiences in cell culture and sterile working techniques who want to establish chemotaxis experiments in their lab.

Schedule Day 1

Schedule Day 2

Start at 10 am

Start at 9 am

• Welcome and Introduction

• Talk 2: Overview of Video, Tracking, and Analysis Software

• Hands-on Part 1: Pipetting Methods – Properly Pipetting and Filling the µ-Slide Chemotaxis 3D

• Hands-on Part 4: Using the Tracking and Analysis Software

Lunch

Lunch

• Hands-on Part 2: Cell Preparation and Cell Seeding Using HT-1080 Cells and Collagen Gel Matrices

• Hands-on Part 5: Finishing Tracking and Analysis

• Talk 1: Physics of Chemotaxis • Hands-on Part 3: Preparation and Set-Up of an Overnight Experiment Using Video Microscopy

• Talk 3: Chemotaxis & Migration Assays and Recent Data • Conclusion & Discussion End of Training at around 4 pm

Summary Day 1 (around 5 pm)

ibidi TIP For upcoming dates and times of individual courses and online registration possibilities, please refer to the course page at: www.ibidi.com

184 184

Participation is free of charge. The number of participants is limited to 8. For registrations and further questions please contact us at info@ibidi.de.


ibidi Practical Course: Cell Cultivation Under Perfusion and Live Cell Imaging 2 day Laboratory Course at ibidi Munich / Germany

Topics: Several adherent cell types are exposed to shear stress conditions in vivo, e.g., endothelial cells in a blood vessel. Culturing cells in vitro under perfusion conditions simulates this mechanical stimulus and induces a more physiological behavior. The objective of the course is to give scientists a profound background in cell culture biology and physical basics of perfusion based assays. In the practical part endothelial cells (HUVEC) are cultured under physiological flow conditions.

Target Group: The course is intended for scientists and technical associates with a profound experience in cell culture and sterile working techniques who want to establish perfusion based assays in their lab.

Schedule Day 1

Schedule Day 2

Start at 10 am

Start at 9 am

• Welcome and Introduction

• Hands-on Part 3: Microscopy of Cell Experiments

• Talk 1: Physics of Flow Chambers

Lunch • Hands-on Part 1: Handling of Channel Slides, Cell Seeding, Medium Exchange • Talk 2: Flow Types and Introduction to the ibidi Pump System • Hands-on Part 2: Exposing Cells to Flow Summary Day 1 (around 5 pm) ibidi TIP For upcoming dates and times of individual courses and online registration possibilities, please refer to the course page at: www.ibidi.com

• Talk 3: Various Experimental Setups and Example Publications, Troubleshooting • Talk 4: Combination of Perfusion Assays and Impedance Measurements Lunch • Hands-on Part 4: Setup of ibidi Pump System and PumpControl Software, Cycle Programming, Flow Rate Measurements, Calibrations • Conclusion & Discussion

ibidi Courses

• Example Calculations and Introduction to the Practical Session

End of Training at around 4 pm

Participation is free of charge. The number of participants is limited to 8. For registrations and further questions please contact us at info@ibidi.de.

185 185


ibidi TIP Watch out for upcoming live webinars or listen to one of ibidi’s previously recorded webinars on www.ibidi.com.

ibidi Webinars ibidi offers web-based seminars on various scientific topics. Register for a live Webinar and hear a live talk about the cutting-edge technologies of your choice followed by a Q&A session. Alternatively, you can listen to one of our previously recorded Webinars.

Cell Cultivation Under Flow - Introduction Several adherent cell types are exposed to shear stress conditions in vivo, e.g., endothelial cells in a blood vessel. This webinar provides an overview of various perfusion applications and experimental setups. The physical background of flow systems will be explained as well as the impact of these factors on cells and the choice of experimental setups.

Cell Cultivation Under Flow – New Insights and Applications This webinar will provide an overview of the function of endothelium as a barrier between the blood stream and the tissue beneath. It will introduce various perfusion applications and experimental setups. In addition, the physical background of flow systems will be explained, as well as the impact of these factors on cells. There will be a special focus on new applications that use the ibidi Pump System, such as impedance measurements, pulsatile flow, and the addition of substances to the flow system.

Tube Formation Assay for Studying the Effect of Drugs on Angiogenesis The webinar provides a detailed overview on sample preparation, image capture and analysis, as well as setting out available methods to interpret data for angiogenesis investigation.

Cell Migration, Wound Healing, and Invasion: Performing an Assay and Quantitative Image Analysis

ibidi Support

This webinar introduces a new method for wound healing and migration assays based on the ibidi Culture-Insert. Wound healing images are analyzed, evaluated and discussed using the WimScratch image analysis tool. Combining both products strongly enhances reproducibility of results.

New Opportunities to Generate and Automatically Quantify Functional Chemotaxis Assays All aspects of chemotaxis assays using µ-Slides Chemotaxis are presented in this webinar: i.e., cell preparation, gradient establishment, video microscopy, image analysis, and data interpretation. Special emphasis is placed on a new automated solution for cell tracking in phase contrast image stacks.

Principles and Basics of Light Microscopy for Cell-Based Assays Cell-based assays use light microscopy as one of the most dominant parameters in the visualization of multi-parameter data sets. This webinar presents the basics of microscopy including the optical properties of glassware and Petri dishes. Finally, you will learn about the principles of fluorescence microscopy techniques and modern live cell imaging, using the specially designed ibidi µ-Slides and µ-Dishes.

LifeAct Actin Marker: A New Tool for Visualization of Dynamic Cellular Processes This webinar deals with the novel actin marker “LifeAct”. It focuses on the principles of cellular processes visualization to investigate the role of actin and its underlying mechanisms. A comparison of LifeAct to other known actin markers such as phalloidin and actin-EGFP completes the seminar.

186 186


ibidi Reference Database: ibidi’s reference database contains more than 3000 peer-reviewed scientific publications. They cite the use of ibidi products in various contexts. For a reference search to find keywords, applications, or products, please visit http://ibidi.com/support/references/.

Cell Mechanics B. Meier, et al., Chemotactic Cell Trapping in Controlled Alternating Gradient Fields, PNAS, 2011

Shear Stress and Cells under Flow C. Boehlke, et al., Primary Cilia Regulate mTORC1 Activity and Cell Size through Lkb1, Nature Cell Biology, 2010

Actin Visualization

Wound Healing

C. Wu, et al.., Arp2/3 is Critical for Lamellipodia and Response to Extracellular Matrix Cues but is Dispensable for Chemotaxis, Cell, 2012

M. Caesar, et al., Leucinerich repeat kinase 2 functionally interacts with microtubules and kinasedependently modulates cell migration, Neurobiology of Disease, 2013

Webinars and References

The following, selected scientific publications cite the use of ibidi products in various contexts:

187 187


Subject Index µ-Dish . . . . . . . . . . . . . . . . . . . 60 µ-Galaxy Incubator . . . . . . . . 127 µ-Plate Angiogenesis 96 well . 86, 101 µ-Plate Family . . . . . . . . . . . . . 88 µ-Slide . . . . . . . . . . . . . . . 66, 176 µ-Slide 18 well - flat . . . . . . . . . 84 µ-Slide 2 well | 4 well | 8 well . . 66, 68 µ-Slide 2 x 9 well . . . . . . . . . . . 70 µ-Slide Angiogenesis . . . . . . 100 µ-Slide Chemotaxis 2D / 3D . . . 109 µ-Slide Click Rack . . . . . . . . . . 29 µ-Slide I . . . . . . . . . . . . . . . . . 71 µ-Slide I Luer Family . . . . . . . 128 µ-Slide III . . . . . . . . . . . . . . . . 130 µ-Slide III 3in1 . . . . . . . . . . . . . . . 111 µ-Slide Ph+ . . . . . . . . . . . . . . . 67 µ-Slide Rack . . . . . . . . . . . . . . 29 µ-Slide VI . . . . . . . . . . . . 78, 130 µ-Slide y-shaped . . . . . . . . . . 132 12 well Chamber . . . . . . . . . . . 85 24 well plate . . . . . . . . . . . . . . 89 2D Migration . . . . . . . . . . 92, 104 2-Photon Microscopy . . . . . . 179 384 well plate . . . . . . . . . . . . . 89 3D cell culture . . 58, 83, 100, 116 3D cell microscopy . . . . . . . . . 58 3D Migration . . . . . . . . . . . . . 104 96 well plate . . . . . . . . . . . . . . 89

ibidi Support

A Abbe number . . . . . . . . . . . . . 171 Actin . . . . . . . . . . . . . . . . . . . . 30 Actin marker . . . . . . . . . . . . . . 31 Actin staining . . . . . . . . . . . . . . 30 Adenovirus . . . . . . . 39, 40, 47-49 Adenovirus cloning . . . . . . . . . 48 Adenovirus purification . . . . . . 49 Adenovirus transduction . . 40, 47 Adipose tissue . . . . . . . . . . . . 166 All-in-one chamber . . . . . 76, 176 Angiogenesis . . . . . . . . . . . . . . 98 Anti-Evaporation oil . . . . . . . . . 53 Aortic ring . . . . . . . . . . . . . . . 153 Application Notes . . . . . . . . . 182 Autofluorescence . . . . . . . . . . 171 Automated image analysis . . . 151

B Barrier function . . . . . . . . . . . 140 Birefringence . . . . . . . . . . . . . 173 Bottom materials . . . . . . . . . . 172 Branching point . . . . . . . 152, 158

188 188

C Ca imaging . . . . . . . . . . . . . . 116 Cell culture geometry . . . . . . 176 Cell culture surfaces . . . . . . . 172 Cell line services . . . . . . . . . . . 36 Cell migration . . . . . 92, 104, 139 Cell tracking . . . . . . . . . . 104, 154 Cell-based assays . . . . . . . . 57, 91 Cell-covered area . . . . . . 93, 156 Chambered coverslip . . . . . . . 66 Channel slide . . . . . . . . . 128, 176 Chemotaxis . . . . . . . . . . . . . . 104 Chemotaxis and Migration Tool . 155 Chemotaxis Assay . . . . . . . . . 104 CO2 Bicarbonate buffer . . . . . . 12 Coating . . . . . . . . . . . . . . . . . 173 Co-cultivation . . . . . . . . . . . . . 58 Collagen . . . . . . . . . . . . . . . . . . 52 Collagen gel . . . . . . . . . . . . . . 105 Collagen IV . . . . . . . . . . . . . . 173 Comet assay . . . . . . . . . . . . . 160 Condensation . . . . . . . . . . 14, 18 Confocal microscopy . . . . . . . 178 Continuous, unidirectional flow . . . . . . . . . . . . . . . . . . . . 114, 118 Courses . . . . . . . . . . . . . 184, 185 Coverslip . . . . . . . . . . . . . . . . 172 CPOx beads . . . . . . . . . . . . . . 25 Culture-Insert . . . . . . . . . . . . . 94 Custom specific slide . . . . . . 149 Cytoskeleton . . . . . . . . . . . . . . 30 DIC Differential Interference Contrast . . . . . . . . . . . . . . . . . 178 2+

FLIM Fluorescence Lifetime Imaging Microscopy . . . . 26, 179 Flow accessories . . . . . . . . . . 133 Flow assay . . . . . . . . . . . . 116, 120 Flow culture . . . . . . . . . . . . . . . 116 Fluidic Unit . . . . . . . . . . . . . . . . 119 Focus stability . . . . . . . . . . . . . 14 Forward migration index . 154, 155 FRAP Fluorescence Recovery After Photobleaching . . . . . . . 179 Freezing medium . . . . . . . . . . . 54 FRET Förster Resonance Energy Transfer . . . . . . . . . . . 179 Functional cell-based assay . . 91 Fuse-It . . . . . . . . . . . . . . . . . . 42

G Gas incubation . . . . . . . . . . . . . 22 Gel matrix . . . . . . . . . . . . 52, 109 Glass bottom . . . . 62, 65, 68, 172 Gradient . . . . . . . . . . . . . . . . . 105 Grid . . . . . . . . . . . . . . . . . . . . . 64

H Heated plate . . . . . . . . . . . . . . 17 Heating insert . . . . . . . . . . . . . 17 High resolution microscopy . . 170 HT-1080 . . . . . . . . . . . . . . 34, 104 Humidifying Chamber Olaf . . . 29 Humidifying column . . . . . . . . 16 Humidity . . . . . . . . . . . . . . . . . 12 HUVEC . . . . . . 107, 118, 122, 123

I D DIC Lid . . . . . . . . . . . . . . . . 62, 68

E ECIS Electric Cell Substrate Impedance Sensing . . . . . . . . 142 ECIS flow assay . . . . . . . 140, 146 Elastically supported surface (ESS) . . . . . . . . . . . . . . . . 63, 174 Elasticity . . . . . . . . . . . . . . . . . 174 Electrode µ-Slide . . . . . . . . . . 148 Endothelial cells . . . . . . . . 114, 123 ESS . . . . . . . . . . . . . . . . . 63, 174 Evaporation . . . . . . . . . . . . 12, 53

F F-Actin . . . . . . . . . . . . . . . . . . . 30 Fibronectin . . . . . . . . . . . . . . . 173

ibidi Gas Incubation System . . 22 ibidi Glass Bottom . . . . . . . . . 172 ibidi Heating System . . 15, 18, 20 ibidi OPAL – Optical O2 Measurement System . . . . . . . 24 ibidi Pump System . . . . . . . . . 118 ibidi Service . . . . . . . . . . . . . . . 36 ibidi Standard Bottom . . . . . . 172 ibiTreat . . . . . . . . . . . . . . . . . . 172 Image analysis . . . . . . . . . . . . . 151 Immersion oil . . . . . . . . . . . . . . 55 Immunofluorescence . . . . . . . 74 Impedance . . . . . . . . . . . . . . 138 Incubator . . . . . . . . . . . . . . . . 127 Interstitial flow . . . . . . . . . . . . . 116 Invasion . . . . . . . . . . . . . . 94, 108 Invasion Assay . . . . . . . . . 94, 108 Inverted microscope . . . . . . . 177


L Lentivirus . . . . . . . . . . . . . . . . . 39 LifeAct . . . . . . . . . . . . . . . . . . . 32 Lipid droplet . . . . . . . . . . . . . . 165 Live cell imaging . . . . . . . . . . . 12 Luer connector . . . . . . . . . . . 133

M Magnification . . . . . . . . . . . 14, 170 Material dispersion . . . . . . . . . 171 Matrigel . . . . . . 98, 100, 101, 109 Membrane fusion . . . . . . . . 41, 42 Membrane labeling . . . . . . . 41, 42 Meniscus . . . . . . . . . . . . . . 67, 177 Metastatic potential . . . . . . . . 141 micro-Insert 4 well . . . . . . . . . 72 Micromanipulation . . . . 59, 61, 62 Microscope, inverted . . . . . . 177 Microscope, upright . . . . . . . 177 Microscopy rack . . . . . . . . . . . 28 Microscopy techniques . . . . . 178 Migration . . . . . . . . . . . . . 92, 104 MM2 fluorophore . . . . . . . . . . 26 Mounting medium . . . . . . . . . . 55 Multi-well plate . . . . . . . . . . . . 88

N NanO2 fluorophore . . . . . . 25, 27 Neurite outgrowth . . . . . . . . . 162 Non-uniform, laminar shear stress . . . . . . . . . . . . . . . . . . . . 115 Numerical aperture . . . . . . . . 170

O2 Control . . . . . . . . . . . . . 13, 24 O2 Measurement . . . . . . . . 24, 26 Olaf . . . . . . . . . . . . . . . . . . . . . 29 OPAL – Optical O2 Measurement System . . . . . . . . . . . . . . . . . . 24 Oscillating flow . . . . . . . . . . . . 115 Oxygen . . . . . . . . . . . . . . . 13, 24

P Perfusion . . . . . . . . . . . . . . . . . 114 Perfusion assay . . . . . . . . 116, 120 Perfusion Set . . . . . . . . . . 119, 124 Peristaltic pump . . . . . . . . . . . 117 Petri dish . . . . . . . . . . . . . . . . . 60 Phalloidin . . . . . . . . . . . . . . . . . 30 Phase contrast . . . . . . . . . 67, 177 Plasmid . . . . . . . . . . . . . . . . . . 33 Poly-D-Lysine . . . . . . . . . . . . 173

Transmission . . . . . . . . . . . . . . 171 Tube formation . . . . 98, 100, 152 Tube length . . . . . . . . . . 100, 152 Tubing . . . . . . . . . . . . . . . . . . 135 TUNEL assay . . . . . . . . . . . . . 167 Turbulent flow . . . . . . . . . . . . . 115

U Q Quantitative image analysis . . 151

Unidirectional, laminar flow . . . 114 Upright microscope . . . . . . . . 177

R

V

Reference database . . . . . . . 187 References . . . . . . . . . . . . . . 187 Refractive index . . . . . . . . . . . 170 Relocation grid . . . . . . . . . . . . 64 Removable chamber . . 72, 85, 94 Resolution . . . . . . . . . . . . . 14, 170 Rolling and adhesion assay . . . 116

Viral cloning . . . . . . . . . . . . . . . 48 Viral vector . . . . . . .33, 39, 40, 48 Virus purification . . . . . . . . . . . 49

S Scratch assay . . . . . . . . . . . . . 94 Screening . . . . . . . . . . . . . . . . 86 Shear rate . . . . . . . . . . . . 114, 124 Shear stress . . . . . . . . . . 114, 124 Silicone insert . . . . . . . . . . 72, 94 Silicone oil . . . . . . . . . . . . . . . . 53 Silicone tubing . . . . . . . . . . . . 135 Spheroids . . . . . . . . 58, 100, 153 Sprouting . . . . . . . . . . . . 100, 153 Stable cell line . . . . . . . . . . 34, 36 Stage-Top incubator . . 15, 18, 20 Standard bottom . . . . . . . . . . 172 Static culture . . . . . . . . . 122, 123 Stem cells . . . . . . . . . . . . . 59, 72 sticky-Slide . . . . . 69, 79, 110, 129 Stop flow experiment . . . . . . . 116 Super-Resolution . . . . . . . . . . 179 Syringe pump . . . . . . . . . 117, 126

W Webinars . . . . . . . . . . . . . . . . 186 Wide-Field Fluorescence . 27, 178 WimAdipose . . . . . . . . . . . . . 166 WimCAM . . . . . . . . . . . . . . . 158 WimColony . . . . . . . . . . . . . . 161 WimComet . . . . . . . . . . . . . . 160 WimCounting . . . . . . . . . . . . 157 WimCytotoxicity . . . . . . . . . . 159 WimLipid . . . . . . . . . . . . . . . . 165 WimNeuron . . . . . . . . . . . . . . 162 WimRetina . . . . . . . . . . . . . . . 163 WimScratch . . . . . . . . . . . 94, 156 WimSprout . . . . . . . . . . 100, 153 WimTaxis . . . . . . . . . . . . 108, 154 WimTransfection . . . . . . . 50, 164 WimTube . . . . . . . . . . . . 100, 152 WimTUNEL . . . . . . . . . . . . . . 167 Workshops . . . . . . . . . . 184, 186 Wound healing . . . . . 92, 94, 156 Wounding . . . . . . . . . 92, 94, 156

T TempControl software . . . . . . 16 Temperature stability . . . . . . 12, 16 TIRF Total Internal Reflection Fluorescence . . . . . . . . . . . . . 178 Tissue culture treated . . . . . . 172 Torpedo DNA . . . . . . . . . . . . . . . 50 Torpedo siRNA . . . . . . . . . . . . . . . 51 Toxicological screening . . . . . . 141 Trajectory plot . . . . . . . . . 107, 155 Transduction . . . . . . . . 33, 40, 47 Transfection . . 33, 40, 50, 51, 164

Cover Cell Photo

Subject Index

O

Poly-L-Lysine . . . . . . . . . . . . . 173 Practical Courses . . . . . . . . . . 184 Proteofection . . . . . . . . . . . . . . 43 Protocols . . . . . . . . . . . . . . . . 182 Pulsatile laminar flow . . . . . . . 114 Pump System . . . . . . . . . . . . . 118 PumpControl software . . . . . . 119

HeLa cells were transiently transfected with the actin plasmid pCAGLifeAct-TagGFP2 (white) via Torpedo DNA. The cells were fixed, permeabilized, and challenged with DAPI (blue). (Courtesy of James J. Faust and David G. Capco, Arizona State University, Tempe, USA)

189 189


Catalog Number Index

Cat. No. Product Description 10974 10976 10977

Cat. No. Product Description 10802 10811 10812 10820 10821 10822 10823 10824 10825 10826 10827 10828 10829 10830 10831 10840 10841 10842 10902 10903 10904 10905 10908 10915

10918

10926

10927

10928 10932

10933

ibidi Support

10934

10936 10937

10940 10961

10962

10963

10964

10965

10966

190 190

10971 10972

Elbow Luer connector Coverslips for 12 well Chamber, removable Coverslips for sticky-Slides In-line Luer injection port Hose Clip Luer Plug Male Female Luer Lock Coupler Luer Connector Male Luer Lock Connector Female Luer Lock Connector Male Y Tube Fitting 0.8 mm ID Y Tube Fitting 1.6 mm ID Fitting Reducer 0.5 to 1.6 Serial Connector for µ-Slides Tube Adapter Set Silicone Tubing 0.5 mm ID Silicone Tubing 0.8 mm ID Silicone Tubing 1.6 mm ID ibidi Pump System Fluidic Unit Fluidic Unit Quad ibidi Pump Notebook ibidi Heating System, Universal Fit, for 1 Chamber, without Heated Lid ibidi Heating System, Universal Fit, for 1 Chamber, with Heated Lid ibidi Heating System, Multi-Well Plates on a Nikon TI-S-ER Motorized Stage ibidi Heating System, Universal Fit, for 4 µ-Slides, with Heated Lid Heated Plate in Multi-Well Format for 4 µ-Slides Heating Insert for ibidi Heating System, Universal Fit, for µ-Dish 35 mm, low Heating Insert for ibidi Heating System, Universal Fit, for all ibidi µ-Slides Heating Insert for ibidi Heating System, Universal Fit, for µ-Dish 35 mm, high Heating Insert Adapter for perfusion assays Heating Insert for ibidi Heating System, Universal Fit, for LabTek™ / LabTek™ II chambered coverglass Syringe Pump KDS 100 Perfusion Set BLUE, length 15 cm, ID 0.8 mm, 10 ml reservoirs Perfusion Set RED, length 15 cm, ID 1.6 mm, 10 ml reservoirs Perfusion Set WHITE, length 50 cm, ID 0.8 mm, 10 ml reservoirs Perfusion Set YELLOW-andGREEN, length 50 cm, ID 1.6 mm, 10 ml reservoirs Perfusion Set YELLOW, length 15 cm, ID 0.5 mm, 2 ml reservoirs Perfusion Set BLACK, length 50 cm, ID 0.5 mm, 2 ml reservoirs Filter/Reservoir Set, 10 ml Filter/Reservoir Set, 2 ml

Page 133 85 69 134 134 134 134 133 133 133 135 135 135 135 134 135 135 135 118 119 119 119 119 18

18

10978 10991 11920 11922 11929 11956 11957 11958

11959 11960

11967

11968

11969

11970 11971

20

11972

18

11980

18

3000150

18

30001100

18

30001200

18

30001500

19

300011000

18

126 124

124

125

125

125

125

121 121

300015000 3000110000 3000120000 3000150000 30001100000 30002200 30002500 300021000

Filter/Reservoir Set, 50 ml Reservoir Holder for Fluidic Unit, 10 ml Reservoir Holder for Fluidic Unit, 2 ml Reservoir Holder for Fluidic Unit, 50 ml µ-Galaxy cell culture incubator ibidi Gas Incubation System for CO2 ibidi Gas Incubation System for CO2 and O2 Air Pressure Generator Syringe Pump Legato 200, Infuse only, 2 syringes Syringe Pump Legato 210, Infuse/Withdraw, 2 syringes Syringe Pump Legato 210 P, Infuse/Withdraw programmable, 2 syringes Syringe Pump Legato 270, Push/Pull, 4 syringes Syringe Pump Legato 270 P, Push/Pull programmable, 4 syringes Syringe Pump Legato 111, Infuse/Withdraw programmable, 2 syringes Syringe Pump Legato 110, Infuse/Withdraw programmable, 1 syringe Syringe Pump Legato 180, Infuse/Withdraw programmable, 2 syringes Syringe Pump Legato 100, Infuse only, 1 syringe Syringe Pump Legato 101, Infuse only, 2 syringes Syringe Pump Legato 130, Infuse/Withdraw, 1 syringe ADAGIO Software for Legato Pumps WimTube Quantitative Tube Formation Image Analysis, 50 images WimTube Quantitative Tube Formation Image Analysis, 100 images WimTube Quantitative Tube Formation Image Analysis, 200 images WimTube Quantitative Tube Formation Image Analysis, 500 images WimTube Quantitative Tube Formation Image Analysis, 1,000 images WimTube Quantitative Tube Formation Image Analysis, 5,000 images WimTube Quantitative Tube Formation Image Analysis, 10,000 images WimTube Quantitative Tube Formation Image Analysis, 20,000 images WimTube Quantitative Tube Formation Image Analysis, 50,000 images WimTube Quantitative Tube Formation Image Analysis, 100,000 images WimScratch Quantitative Wound Healing Image Analysis, 200 images WimScratch Quantitative Wound Healing Image Analysis, 500 images WimScratch Quantitative Wound Healing Image Analysis, 1,000 images

Page 121 121 121 121 127 22 22 22 126 126 126

126 126

126

126

126

126 126 126 126 152

152

152

152

152

152

152

152

152

152

156

156

156

Cat. No. Product Description 30002- WimScratch Quantitative 5000 Wound Healing Image Analysis, 5,000 images 30002- WimScratch Quantitative 10000 Wound Healing Image Analysis, 10,000 images 30002- WimScratch Quantitative 20000 Wound Healing Image Analysis, 20,000 images 30002- WimScratch Quantitative 50000 Wound Healing Image Analysis, 50,000 images 30002- WimScratch Quantitative 100000 Wound Healing Image Analysis, 100,000 images 30003- WimTaxis Quantitative 10 Chemotaxis Cell Tracking Image Analysis, 10 videos 30003- WimTaxis Quantitative 50 Chemotaxis Cell Tracking Image Analysis, 50 videos 30003- WimTaxis Quantitative 100 Chemotaxis Cell Tracking Image Analysis, 100 videos 30003- WimTaxis Quantitative 200 Chemotaxis Cell Tracking Image Analysis, 200 videos 30003- WimTaxis Quantitative 500 Chemotaxis Cell Tracking Image Analysis, 500 videos 30003- WimTaxis Quantitative 1000 Chemotaxis Cell Tracking Image Analysis, 1000 videos 30004- WimSprout Quantitative 50 Sprouting Spheroid Assay Image Analysis, 50 images 30004- WimSprout Quantitative 100 Sprouting Spheroid Assay Image Analysis, 100 images 30004- WimSprout Quantitative 200 Sprouting Spheroid Assay Image Analysis, 200 images 30004- WimSprout Quantitative 500 Sprouting Spheroid Assay Image Analysis, 500 images 30004- WimSprout Quantitative 1000 Sprouting Spheroid Assay Image Analysis, 1,000 images 30004- WimSprout Quantitative 5000 Sprouting Spheroid Assay Image Analysis, 5,000 images 30004- WimSprout Quantitative 10000 Sprouting Spheroid Assay Image Analysis, 10,000 images 30004- WimSprout Quantitative 20000 Sprouting Spheroid Assay Image Analysis, 20,000 images 30004- WimSprout Quantitative 50000 Sprouting Spheroid Assay Image Analysis, 50,000 images 30004- WimSprout Quantitative 100000 Sprouting Spheroid Assay Image Analysis, 100,000 images 30005- WimCounting Quantitative 200 Cell Counting Image Analysis, 200 images 30005- WimCounting Quantitative 500 Cell Counting Image Analysis, 500 images 30005- WimCounting Quantitative 1000 Cell Counting Image Analysis, 1,000 images 30005- WimCounting Quantitative 5000 Cell Counting Image Analysis, 5,000 images 30005- WimCounting Quantitative 10000 Cell Counting Image Analysis, 10,000 images 30005- WimCounting Quantitative 20000 Cell Counting Image Analysis, 20,000 images

Page 156

156

156

156

156

154

154

154

154

154

154

153

153

153

153

153

153

153

153

153

153

157

157

157

157

157

157


Cat. No. Product Description

Page 157

157

158

158

158

158

158

158

158

158

159

159

159

159

159

159

159

159

160

160

160

160

160

160

160

Cat. No. Product Description 30008- WimComet Quantitative 100000 Comet Assay Image Analysis, 100,000 images 30009- WimColony Quantitative 500 Colony Forming Image Analysis, 500 images 30009- WimColony Quantitative 1000 Colony Forming Image Analysis, 1,000 images 30009- WimColony Quantitative 2000 Colony Forming Image Analysis, 2,000 images 30009- WimColony Quantitative 5000 Colony Forming Image Analysis, 5,000 images 30009- WimColony Quantitative 10000 Colony Forming Image Analysis, 10,000 images 30009- WimColony Quantitative 20000 Colony Forming Image Analysis, 20,000 images 30009- WimColony Quantitative 50000 Colony Forming Image Analysis, 50,000 images 30009- WimColony Quantitative 100000 Colony Forming Image Analysis, 100,000 images 30013- WimTaxis Quantitative 10 Chemotaxis Bacteria Tracking Image Analysis, 10 videos 30013- WimTaxis Quantitative 50 Chemotaxis Bacteria Tracking Image Analysis, 50 videos 30013- WimTaxis Quantitative 100 Chemotaxis Bacteria Tracking Image Analysis, 100 videos 30013- WimTaxis Quantitative 200 Chemotaxis Bacteria Tracking Image Analysis, 200 videos 30013- WimTaxis Quantitative 500 Chemotaxis Bacteria Tracking Image Analysis, 500 videos 30013- WimTaxis Quantitative 1000 Chemotaxis Bacteria Tracking Image Analysis, 1,000 videos 30014- WimSprout Quantitative 50 Aortic Ring Assay Image Analysis, 50 images 30014- WimSprout Quantitative 100 Aortic Ring Assay Image Analysis, 100 images 30014- WimSprout Quantitative 200 Aortic Ring Assay Image Analysis, 200 images 30014- WimSprout Quantitative 500 Aortic Ring Assay Image Analysis, 500 images 30014- WimSprout Quantitative 1000 Aortic Ring Assay Image Analysis, 1,000 images 30014- WimSprout Quantitative 5000 Aortic Ring Assay Image Analysis, 5,000 images 30014- WimSprout Quantitative 10000 Aortic Ring Assay Image Analysis, 10,000 images 30014- WimSprout Quantitative 20000 Aortic Ring Assay Image Analysis, 20,000 images 30014- WimSprout Quantitative 50000 Aortic Ring Assay Image Analysis, 50,000 images 30014- WimSprout Quantitative 100000 Aortic Ring Assay Image Analysis, 100,000 images 30024- WimSprout Quantitative 50 Fibrin Gel Bead Assay Image Analysis, 50 images 30024- WimSprout Quantitative 100 Fibrin Gel Bead Assay Image Analysis, 1,00 images

Page 160

161

161

161

161

161

161

161

161

154

154

154

154

154

154

153

153

153

153

153

153

153

153

153

153

153

153

Cat. No. Product Description 30024- WimSprout Quantitative 200 Fibrin Gel Bead Assay Image Analysis, 200 images 30024- WimSprout Quantitative 500 Fibrin Gel Bead Assay Image Analysis, 500 images 30024- WimSprout Quantitative 1000 Fibrin Gel Bead Assay Image Analysis, 1,000 images 30024- WimSprout Quantitative 5000 Fibrin Gel Bead Assay Image Analysis, 5,000 images 30024- WimSprout Quantitative 10000 Fibrin Gel Bead Assay Image Analysis, 10,000 images 30024- WimSprout Quantitative 20000 Fibrin Gel Bead Assay Image Analysis, 20,000 images 30024- WimSprout Quantitative 50000 Fibrin Gel Bead Assay Image Analysis, 50,000 images 30024- WimSprout Quantitative 100000 Fibrin Gel Bead Assay Image Analysis, 100,000 images 30100- WimNeuron Quantitative 500 Neurite Outgrowth Image Analysis, 500 images 30100- WimNeuron Quantitative 1000 Neurite Outgrowth Image Analysis, 1,000 images 30100- WimNeuron Quantitative 2000 Neurite Outgrowth Image Analysis, 2,000 images 30100- WimNeuron Quantitative 5000 Neurite Outgrowth Image Analysis, 5,000 images 30100- WimNeuron Quantitative 10000 Neurite Outgrowth Image Analysis, 10,000 images 30100- WimNeuron Quantitative 20000 Neurite Outgrowth Image Analysis, 20,000 images 30100- WimNeuron Quantitative 50000 Neurite Outgrowth Image Analysis, 50,000 images 30100- WimNeuron Quantitative 100000 Neurite Outgrowth Image Analysis, 100,000 images 30101- WimRetina Quantitative 500 Retina Vessels Image Analysis, 500 images 30101- WimRetina Quantitative 1000 Retina Vessels Image Analysis, 1,000 images 30101- WimRetina Quantitative 2000 Retina Vessels Image Analysis, 2,000 images 30101- WimRetina Quantitative 5000 Retina Vessels Image Analysis, 5,000 images 30101- WimRetina Quantitative 10000 Retina Vessels Image Analysis, 10,000 images 30101- WimRetina Quantitative 20000 Retina Vessels Image Analysis, 20,000 images 30101- WimRetina Quantitative 50000 Retina Vessels Image Analysis, 50,000 images 30101- WimRetina Quantitative 100000 Retina Vessels Image Analysis, 100,000 images 30102- WimTransfection Quantitative 500 Transfection Efficiency Image Analysis, 500 images 30102- WimTransfection Quantitative 1000 Transfection Efficiency Image Analysis, 1,000 images 30102- WimTransfection Quantitative 2000 Transfection Efficiency Image Analysis, 2,000 images

Page 153

153

153

153

153

153

153

153

162

162

162

162

162

162

162

162

163

163

163

163

163

163

163

163

164

Catalog Number Index

30005- WimCounting Quantitative 50000 Cell Counting Image Analysis, 50,000 images 30005- WimCounting Quantitative 100000 Cell Counting Image Analysis, 100,000 images 30006- WimCAM Quantitative 500 Chorioallantoic Membrane Assay Image Analysis, 500 images 30006- WimCAM Quantitative 1000 Chorioallantoic Membrane Assay Image Analysis, 1,000 images 30006- WimCAM Quantitative 2000 Chorioallantoic Membrane Assay Image Analysis, 2,000 images 30006- WimCAM Quantitative 5000 Chorioallantoic Membrane Assay Image Analysis, 5,000 images 30006- WimCAM Quantitative 10000 Chorioallantoic Membrane Assay Image Analysis, 10,000 images 30006- WimCAM Quantitative 20000 Chorioallantoic Membrane Assay Image Analysis, 20,000 images 30006- WimCAM Quantitative 50000 Chorioallantoic Membrane Assay Image Analysis, 50,000 images 30006- WimCAM Quantitative 100000 Chorioallantoic Membrane Assay Image Analysis, 100,000 images 30007- WimCytotoxicity Quantitative 500 Cytotoxicity Image Analysis, 500 images 30007- WimCytotoxicity Quantitative 1000 Cytotoxicity Image Analysis, 1,000 images 30007- WimCytotoxicity Quantitative 2000 Cytotoxicity Image Analysis, 2,000 images 30007- WimCytotoxicity Quantitative 5000 Cytotoxicity Image Analysis, 5,000 images 30007- WimCytotoxicity Quantitative 10000 Cytotoxicity Image Analysis, 10,000 images 30007- WimCytotoxicity Quantitative 20000 Cytotoxicity Image Analysis, 20,000 images 30007- WimCytotoxicity Quantitative 50000 Cytotoxicity Image Analysis, 50,000 images 30007- WimCytotoxicity Quantitative 100000 Cytotoxicity Image Analysis, 100,000 images 30008- WimComet Quantitative 500 Comet Assay Image Analysis, 500 images 30008- WimComet Quantitative Comet Assay Image Analysis, 1000 1,000 images 30008- WimComet Quantitative 2000 Comet Assay Image Analysis, 2,000 images 30008- WimComet Quantitative 5000 Comet Assay Image Analysis, 5,000 images 30008- WimComet Quantitative 10000 Comet Assay Image Analysis, 10,000 images 30008- WimComet Quantitative 20000 Comet Assay Image Analysis, 20,000 images 30008- WimComet Quantitative 50000 Comet Assay Image Analysis, 50,000 images

164

164

191 191


ibidi Support

Cat. No. Product Description

192 192

30102- WimTransfection Quantitative 5000 Transfection Efficiency Image Analysis, 5,000 images 30102- WimTransfection Quantitative 10000 Transfection Efficiency Image Analysis, 10,000 images 30102- WimTransfection Quantitative 20000 Transfection Efficiency Image Analysis, 20,000 images 30102- WimTransfection Quantitative 50000 Transfection Efficiency Image Analysis, 50,000 images 30102- WimTransfection Quantitative 100000 Transfection Efficiency Image Analysis, 100,000 images 30103- WimAdipose Quantitative 500 Adipose Tissue Image Analysis, 500 images 30103- WimAdipose Quantitative 1000 Adipose Tissue Image Analysis, 1,000 images 30103- WimAdipose Quantitative 2000 Adipose Tissue Image Analysis, 2,000 images 30103- WimAdipose Quantitative 5000 Adipose Tissue Image Analysis, 5,000 images 30103- WimAdipose Quantitative 10000 Adipose Tissue Image Analysis, 10,000 images 30103- WimAdipose Quantitative 20000 Adipose Tissue Image Analysis, 20,000 images 30103- WimAdipose Quantitative 50000 Adipose Tissue Image Analysis, 50,000 images 30103- WimAdipose Quantitative 100000 Adipose Tissue Image Analysis, 100,000 images 30104- WimLipid Quantitative Lipid 500 Droplet Image Analysis, 500 images 30104- WimLipid Quantitative Lipid 1000 Droplet Image Analysis, 1,000 images 30104- WimLipid Quantitative Lipid 2000 Droplet Image Analysis, 2,000 images 30104- WimLipid Quantitative Lipid 5000 Droplet Image Analysis, 5,000 images 30104- WimLipid Quantitative Lipid 10000 Droplet Image Analysis, 10,000 images 30104- WimLipid Quantitative Lipid 20000 Droplet Image Analysis, 20,000 images 30104- WimLipid Quantitative Lipid 50000 Droplet Image Analysis, 50,000 images 30104- WimLipid Quantitative Lipid 100000 Droplet Image Analysis, 100,000 images 30105- WimTUNEL Quantitative 500 TUNEL Assay Image Analysis, 500 images 30105- WimTUNEL Quantitative 1000 TUNEL Assay Image Analysis, 1,000 images 30105- WimTUNEL Quantitative 2000 TUNEL Assay Image Analysis, 2,000 images 30105- WimTUNEL Quantitative 5000 TUNEL Assay Image Analysis, 5,000 images 30105- WimTUNEL Quantitative 10000 TUNEL Assay Image Analysis, 10,000 images 30105- WimTUNEL Quantitative 20000 TUNEL Assay Image Analysis, 20,000 images

Page 164

164

164

164

164

166

166

166

166

166

166

166

166

165

165

165

165

165

165

165

165

167

167

167

167

167

167

Cat. No. Product Description

Page

30105- WimTUNEL Quantitative 167 50000 TUNEL Assay Image Analysis, 50,000 images 30105- WimTUNEL Quantitative 167 100000 TUNEL Assay Image Analysis, 100,000 images 40101 HT-1080 LifeAct-TagGFP2 34 40102 HT-1080 LifeAct-TagRFP 34 50001 ibidi Mounting Medium 55 50051 ibidi Anti-Evaporation Oil 53 50101 ibidi Immersion Oil 55 50201 Collagen Type I, 5 mg/ml, 1 x 5 ml 52 50202 Collagen Type I, 5 mg/ml, 4 x 5 ml 52 52 50203 Collagen Type I, 5 mg/ml, 1 x 100 ml 52 50204 Collagen Type I, 10 mg/ml, 1 x 5 ml 52 50205 Collagen Type I, 10 mg/ml, 4 x 5 ml 52 50206 Collagen Type I, 10 mg/ml, 1 x 100 ml 50301 ibiBoost Adeno Adenovirus 47 Transduction Enhancer 33 60101 Plasmid pCMVLifeAct-TagGFP2 33 60102 Plasmid p CMVLifeAct-TagRFP 33 60106 Plasmid p CAGLifeAct-TagGFP2 33 60107 Plasmid pCAGLifeAct-TagRFP 33 60121 Adenovirus rAVCMV-LifeAct-TagGFP2 33 60122 Adenovirus rAVCMV-LifeAct-TagRFP 60200 Fuse-Itgreen, 100 µl solution 42 60201 Fuse-Itgreen, 400 µl solution 42 60202 Fuse-Itred, 100 µl solution 42 60203 Fuse-Itred, 400 µl solution 42 60204 Fuse-Itdred,100 µl solution 42 60205 Fuse-Itdred, 400 µl solution 42 60206 Fuse-ItIR, 100 µl solution 42 60207 Fuse-ItIR, 400 µl solution 42 60210 Fuse-It-L, lyophilized, 45 for 100 µl solution 60211 Fuse-It-L, lyophilized, 45 for 4 x 25 µl solution 60212 Fuse-It-L, lyophilized, 45 for 400 µl solution 60213 Fuse-It-L, lyophilized, 45 for 4 x 100 µl solution 60220 Fuse-It-P, lyophilized, 43 for 100 µl solution 60221 Fuse-It-P, lyophilized, 43 for 4 x 25 µl solution 60222 Fuse-It-P, lyophilized, 43 for 400 µl solution 60223 Fuse-It-P, lyophilized, 43 for 4 x 100 µl solution 60250 Fuse-It-MP, 100 µl solution 46 60251 Fuse-It-MP, 400 µl solution 46 60260 Fuse-It-T, 100 µl solution 45 60261 Fuse-It-T, 4 x 25 µl solution 45 60320 Fuse-It-B, 100 µl solution 44 60321 Fuse-It-B, 400 µl solution 44 60322 Fuse-It-B, 100 µl solution 44 60323 Fuse-It-B, 400 µl solution 44 60420 Fuse-It-Beads, 100 µl solution 44 60421 Fuse-It-Beads, 400 µl solution 44 50 60610 Torpedo DNA , Transfection reagent, 0.5 ml 50 60611 TorpedoDNA , Transfection reagent, 1.5 ml 50 60612 TorpedoDNA , Transfection reagent, 2 x 2.0 ml 51 60620 TorpedosiRNA, Transfection reagent, 0.5 ml 51 60621 TorpedosiRNA , Transfection reagent, 1.5 ml 51 60622 TorpedosiRNA , Transfection reagent, 2 x 2.0 ml

Cat. No. Product Description 60661

60662

60663 60681 60682 70101 70110 71001 71609 71612 71614 71616 71617 72001 72002 72003 72005 72010 72021 72030 72040 72096 72097 72098 74001 74051 74101 74102 74111 74112 74151

74161

80003 80007 80008 80009 80021 80022 80023 80024 80025 80030 80031 80035 80036 80037 80050 80055 80101 80102 80106 80110 80111

ibiClone Adeno Adenoviral Cloning Kit, pO6A5 shuttle vector for gene overexpression (CMV promoter) ibiClone Adeno Adenoviral Cloning Kit, pO6A5 shuttle vector for silencing (U6 promoter) Consumables ibiClone Adeno Adenoviral Cloning Kit ibiPure Adeno Adenovirus Purification Kit, 12 preps ibiPure Adeno Adenovirus Purification Kit, 24 preps ECIS Flow array channel µ-Slide with 8 electrodes ECIS Flow array 10E channel µ-Slide with 8 x 10 electrodes ECIS Flow Module ECIS Transwell Measurement System ECIS 16 well station ECIS 96 well station ECIS Model Z ECIS Model Z Theta ECIS Cultureware 8W1E PET ECIS Cultureware 8W2X1E, Medusa PET ECIS Cultureware 8W1E DD PET ECIS Cultureware 8W2LE PET ECIS Cultureware 8W10E PET ECIS Cultureware 8W1LE PET ECIS Cultureware 8WCP PET ECIS Cultureware 8W10E+ PET ECIS Cultureware 96W1E+ PET ECIS Cultureware 96W10idf PET ECIS Cultureware 96W20idf PET ibidi OPAL - Optical O2 Measurement System Adapter Set CPOx-Beads, 50 µm, red, 3 mg CPOx-Beads, 50 µm, red, 10 mg CPOx-Beads, 50 µm, orange, 3 mg CPOx-Beads, 50 µm, orange, 10 mg NanO2, fluorescence lifetime probe for intracellular O2 measurement, 100 µg MM2, fluorescence intensity probe for intracellular O2 measurement, 100 µg µ-Slide Rack µ-Slide Click Rack Olaf, humidifying chamber Olaf, humidifying chamber, with µ-Slide click rack ibidi Freezing Medium Classic, 10 ml ibidi Freezing Medium Classic, 5 x 20 ml ibidi Freezing Medium Classic, 120 ml ibidi Freezing Medium Direct, 20 ml ibidi Freezing Medium HRM, 20 ml µ-Slide Microscopy Rack, base Magnetic lid for µ-Slides µ-Dish Microscopy Rack, base Magnetic lid for µ-Dish 35 mm, low Magnetic lid for µ-Dish 35 mm, high DIC Lid für µ-Dishes DIC Lid for µ-Slides µ-Slide I, Collagen IV µ-Slide I, Fibronectin µ-Slide I, ibiTreat, tissue culture treated µ-Slide I, Poly-L-Lysine µ-Slide I, hydrophobic, uncoated

Page 48

48

48 49 49 146 146 143 146 143 143 143 143 144 145 145 145 144 145 145 144 146 146 146 24 24 25 25 25 25 27

27

29 29 29 29 54 54 54 54 54 28 28 28 28 28 62 68 71 71 71 71 71


Cat. No. Product Description 80115 80121 80122 80123 80124 80125 80126 80131 80136 80151 80156 80161 80162 80163 80164 80165 80166 80168 80171 80172 80173 80174 80175 80176 80178 80181 80182 80183 80184 80185 80186 80188 80191

80198 80206 80209 80241 80245 80246 80281 80282 80283 80284 80285 80286 80287 80291 80292 80293 80294 80295 80296 80297

Page 71 132

Cat. No. Product Description 80302 80306

132 132 132 132 132 61

80311 80312 80313 80314 80315 80316

61 80322 64 80326 64 128

80328 80331

128 128 128 128 128

80332 80333 80342 80343 80341

129 128

80406

128 128 128 128 128

80409

129 128 128 128 128 128 128 129 128 128 128 128 128 128 129 95 95 95 73 73 66 66 66 66 66 66 68 67 67 67 67 67 67 68

80421 80422 80423 80424 80425 80426 80427 80441 80442 80443 80444 80445 80446 80447 80601 80602 80603 80604 80605 80606 80608 80621 80622 80623 80624 80625 80626 80661 80662 80663 80664 80665 80666 80821 80822 80823 80824

µ-Slide Chemotaxis 2D, Collagen IV µ-Slide Chemotaxis 2D, ibiTreat, tissue culture treated µ-Slide III 3in1, hydrophobic, uncoated µ-Slide III 3in1, Collagen IV µ-Slide III 3in1, Fibronectin µ-Slide III 3in1, Poly-L-Lysine µ-Slide III 3in1, Poly-D-Lysine µ-Slide III 3in1, ibiTreat, tissue culture treated µ-Slide Chemotaxis 3D, Collagen IV µ-Slide Chemotaxis 3D, ibiTreat, tissue culture treated sticky-Slide Chemotaxis 3D µ-Slide III 0.1, hydrophobic, uncoated µ-Slide III 0.1, Collagen IV µ-Slide III 0.1, Fibronectin µ-Slide III 0.4, Collagen IV µ-Slide III 0.4, Fibronectin µ-Slide III 0.4, hydrophobic, uncoated micro-Insert 4 well in µ-Dish 35 mm, high, ibiTreat, tissue culture treated 25 micro-Inserts 4 well for self-insertion µ-Slide 4 well, hydrophobic, uncoated µ-Slide 4 well, Collagen IV µ-Slide 4 well, Fibronectin µ-Slide 4 well, Poly-L-Lysine µ-Slide 4 well, Poly-D-Lysine µ-Slide 4 well, ibiTreat, tissue culture treated µ-Slide 4 well glass botttom µ-Slide 4 well Ph+, hydrophobic, uncoated µ-Slide 4 well Ph+, Collagen IV µ-Slide 4 well Ph+, Fibronectin µ-Slide 4 well Ph+, Poly-L-Lysine µ-Slide 4 well Ph+, Poly-D-Lysine µ-Slide 4 well Ph+, ibiTreat, tissue culture treated µ-Slide 4 well Ph+ glass botttom µ-Slide VI 0.4, hydrophobic, uncoated µ-Slide VI 0.4, Collagen IV µ-Slide VI 0.4, Fibronectin µ-Slide VI 0.4, Poly-L-Lysine µ-Slide VI 0.4, Poly-D-Lysine µ-Slide VI 0.4, ibiTreat, tissue culture treated sticky-Slide VI 0.4 µ-Slide VI - flat, hydrophobic, uncoated µ-Slide VI - flat, Collagen IV µ-Slide VI - flat, Fibronectin µ-Slide VI - flat, Poly-L-Lysine µ-Slide VI - flat, Poly-D-Lysine µ-Slide VI - flat, ibiTreat, tissue culture treated µ-Slide VI 0.1, hydrophobic, uncoated µ-Slide VI 0.1, Collagen IV µ-Slide VI 0.1, Fibronectin µ-Slide VI 0.1, Poly-L-Lysine µ-Slide VI 0.1, Poly-D-Lysine µ-Slide VI 0.1, ibiTreat, tissue culture treated µ-Slide 8 well, hydrophobic, uncoated µ-Slide 8 well, Collagen IV µ-Slide 8 well, Fibronectin µ-Slide 8 well, Poly-L-Lysine

Page 109

Cat. No. Product Description 80825 80826

109 111 111 111 111 111 111 109 109 110 130 130 130 131 131 131 73

80827 80828 81101 81106 81121 81122 81123 81128 81131 81136 81148 81151 81156 81158 81161 81166

73 81168 66 66 66 66 66 66 68 67 67 67 67 67 67 68 78 78 78 78 78 78 79 80 80 80 80 80 80 130 130 130 130 130 130 66 66 66 66

81176

81191 81201 81291 81391 81501 81506 81531 81801 81802 81803 81804 81805 81806 81821 81822 81823 81824 81825 81826 81831 82401 82406 88401 89621 89626 89646

µ-Slide 8 well, Poly-D-Lysine µ-Slide 8 well, ibiTreat, tissue culture treated µ-Slide 8 well glass botttom sticky-Slide 8 well µ-Slide I Luer variety pack, hydrophobic, uncoated µ-Slide I Luer variety pack, ibiTreat, tissue culture treated µ-Slide I 0.1 Luer, hydrophobic, uncoated µ-Slide I 0.1 Luer, Collagen IV µ-Slide I 0.1 Luer, Fibronectin sticky-Slide I 0.1 Luer µ-Dish 50 mm, low hydrophobic, uncoated µ-Dish 50 mm, low ibiTreat, tissue culture treated µ-Dish 35 mm, high glass bottom Grid-50 µ-Dish 35 mm, high, hydrophobic, uncoated µ-Dish 35 mm, high, ibiTreat, tissue culture treated µ-Dish 35 mm, high glass bottom µ-Dish 35 mm, high Grid-500, hydrophobic, uncoated µ-Dish 35 mm, high Grid-500, ibiTreat, tissue culture treated µ-Dish 35 mm, high glass bottom Grid-500 Culture-Insert in µ-Dish 35 mm, high, ibiTreat, tissue culture treated µ-Dish 35 mm, high ESS, 28 kPa 12 well Chamber, removable µ-Dish 35 mm, high ESS, 1.5 kPa µ-Dish 35 mm, high ESS, 15 kPa µ-Slide Angiogenesis, hydrophobic, uncoated µ-Slide Angiogenesis, ibiTreat, tissue culture treated µ-Slide Angiogenesis, Microdissection µ-Slide 2 x 9 well, hydrophobic, uncoated µ-Slide 2 x 9 well, Collagen IV µ-Slide 2 x 9 well, Fibronectin µ-Slide 2 x 9 well, Poly-L-Lysine µ-Slide 2 x 9 well, Poly-D-Lysine µ-Slide 2 x 9 well, ibiTreat, tissue culture treated µ-Slide 18 well - flat, hydrophobic, uncoated µ-Slide 18 well - flat, Collagen IV µ-Slide 18 well - flat, Fibronectin µ-Slide 18 well - flat, Poly-L-Lysine µ-Slide 18 well - flat, Poly-D-Lysine µ-Slide 18 well - flat, ibiTreat, tissue culture treated µ-Slide 18 well - flat, Microdissection µ-Plate 24 well, hydrophobic, uncoated µ-Plate 24 well, ibiTreat, tissue culture treated µ-Plate 384 well, hydrophobic, uncoated µ-Plate 96 well, hydrophobic, uncoated µ-Plate 96 well, ibiTreat, tissue culture treated µ-Plate Angiogenesis 96 well, ibiTreat, tissue culture treated

Page 66 66 68 69 128 128 128 128 128 129 62 62 65 61 61 62 64 64 65 95

63 85 63 63 100 100 100 70 70 70 70 70 70 84 84 84 84 84 84 84 89 89 89

Catalog Number Index

80192 80193 80194 80195 80196

µ-Slide I, Poly-D-Lysine µ-Slide y-shaped, hydrophobic, uncoated µ-Slide y-shaped, Collagen IV µ-Slide y-shaped, Fibronectin µ-Slide y-shaped, Poly-L-Lysine µ-Slide y-shaped, Poly-D-Lysine µ-Slide y-shaped, ibiTreat, tissue culture treated µ-Dish 35 mm, low hydrophobic, uncoated µ-Dish 35 mm, low ibiTreat, tissue culture treated µ-Dish 35 mm, low Grid-500, hydrophobic, uncoated µ-Dish 35 mm, low Grid-500, ibiTreat, tissue culture treated µ-Slide I 0.2 Luer, hydrophobic, uncoated µ-Slide I 0.2 Luer, Collagen IV µ-Slide I 0.2 Luer, Fibronectin µ-Slide I 0.2 Luer, Poly-L-Lysine µ-Slide I 0.2 Luer, Poly-D-Lysine µ-Slide I 0.2 Luer, ibiTreat, tissue culture treated sticky-Slide I 0.2 Luer µ-Slide I 0.4 Luer, hydrophobic, uncoated µ-Slide I 0.4 Luer, Collagen IV µ-Slide I 0.4 Luer, Fibronectin µ-Slide I 0.4 Luer, Poly-L-Lysine µ-Slide I 0.4 Luer, Poly-D-Lysin µ-Slide I 0.4 Luer, ibiTreat, tissue culture treated sticky-Slide I 0.4 Luer µ-Slide I 0.6 Luer, hydrophobic, uncoated µ-Slide I 0.6 Luer, Collagen IV µ-Slide I 0.6 Luer, Fibronectin µ-Slide I 0.6 Luer, Poly-L-Lysine µ-Slide I 0.6 Luer, Poly-D-Lysine µ-Slide I 0.6 Luer, ibiTreat, tissue culture treated sticky-Slide I 0.6 Luer µ-Slide I 0.8 Luer, hydrophobic, uncoated µ-Slide I 0.8 Luer, Collagen IV µ-Slide I 0.8 Luer, Fibronectin µ-Slide I 0.8 Luer, Poly-L-Lysine µ-Slide I 0.8 Luer, Poly-D-Lysine µ-Slide I 0.8 Luer, ibiTreat, tissue culture treated sticky-Slide I 0.8 Luer Culture-Insert in µ-Dish 35 mm, low 25 Culture-Inserts for self-insertion Culture-Insert 24 micro-Insert 4 well 24, hydrophobic, uncoated micro-Insert 4 well 24, tissue culture treated µ-Slide 2 well, hydrophobic, uncoated µ-Slide 2 well, Collagen IV µ-Slide 2 well, Fibronectin µ-Slide 2 well, Poly-L-Lysine µ-Slide 2 well, Poly-D-Lysine µ-Slide 2 well, ibiTreat, tissue culture treated µ-Slide 2 well glass botttom µ-Slide 2 well Ph+, hydrophobic, uncoated µ-Slide 2 well Ph+, Collagen IV µ-Slide 2 well Ph+, Fibronectin µ-Slide 2 well Ph+, Poly-L-Lysine µ-Slide 2 well Ph+, Poly-D-Lysine µ-Slide 2 well Ph+, ibiTreat, tissue culture treated µ-Slide 2 well Ph+ glass bottom

89 89 101

193 193


194

Notes


Notes

195


196



ibidi GmbH Am Klopferspitz 19 82152 Martinsried (MĂźnchen) Germany

ibidi 53! )NC 510 Charmany Drive, Suite 268 Madison, Wisconsin 53719-1235 USA

Toll free within Germany: Phone: 0800 / 00 11 11 28 Fax: 0800 / 00 11 11 29

Phone: +1 608 441 8181 Fax: +1 608 441 8383

International calls: Phone: +49 89 / 520 46 17 - 0 Fax: +49 89 / 520 46 17 - 59

E-Mail: www.ibidi.com

E-Mail: info@ibidi.com www.ibidi.com

Certified ISO 9001, ISO 13485 All ibidi products are for research use only! Errors and omissions excepted.

Š ibidi GmbH, V 1.2 / 2015 / 04

For free samples, application notes, and handling movies, please visit us at:

www.

.com


Millions discover their favorite reads on issuu every month.

Give your content the digital home it deserves. Get it to any device in seconds.